- Email: [email protected]
Rosiglitazone and pioglitazone aggravate doxorubicin-induced cardiomyopathy in Wistar rats
Biomedicine & Aging Pathology 1 (2011) 65–71
Original article
Rosiglitazone and pioglitazone aggravate doxorubicin-induced cardiomyopathy in Wistar rats Priyadarshan Saraogi , Krishna Kolappa Pillai , Bhulan Kumar Singh , Kiran Dubey ∗ Department of pharmacology, faculty of pharmacy, Jamia Hamdard (Hamdard University), 110062 New Delhi, India
a r t i c l e
i n f o
Article history: Received 18 September 2010 Accepted 6 December 2010 Keywords: Rosiglitazone Cardiomyopathy Thiazolidinediones
a b s t r a c t Recently the cardiovascular safety of thiazolidinediones (TZDs) has been questioned. This study was designed to assess the effect of rosiglitazone (ROSI 5 mg/kg p.o.) and pioglitazone (PIO 10 mg/kg p.o.) on doxorubicin (DOX 15 mg/kg i.p. single dose) induced cardiomyopathy in Wistar rats. DOX (15 mg/kg i.p. single dose) induced cardiomyopathy was evidenced by a significant (p < 0.001) increase in serum lactate dehydrogenase (LDH), cholesterol profile, thiobarbituric acid reactive substance (TBARS), catalase (CAT) levels and a significant (p < 0.001) decrease in glutathione and superoxide dismutase levels followed by histopathological and transmission electron microscopic changes. Rosiglitazone (5 mg/kg p.o.) and pioglitazone (10 mg/kg p.o.) were administered for 14 days and doxorubicin (15 mg/kg i.p. single dose) was injected on the 10th day of drug treatment. The biochemical estimations revealed that treatment with both these drugs significantly (p < 0.001) increased the serum LDH, cardiac TBARS and catalase levels, while the antioxidant enzyme levels of glutathione and superoxide dismutase were reduced. The administration of rosiglitazone significantly (p < 0.001) reduced the level of serum high density lipoprotein (HDL) and raised the level of low density lipoprotein (LDL) while pioglitazone treatment had shown entirely opposite trend; significantly (p < 0.001) increased HDL level and lowered the LDL level. The transmission electron microscopy and histopathology of rat’s cardiac tissue, however, revealed that treatment with both these drugs caused extensive damage to the myocardium as evidenced by condensed chromatin marginates, loss of myofibrils and vacuolization and aggravated doxorubicin-induced cardiomyopathy. © 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a group of syndrome characterized by hyperglycemia, altered metabolism of lipids, carbohydrate, proteins and increased risk of complications from cardiovascular diseases [1]. Thiazolidinediones (TZDs) is a novel class of oral antidiabetic drugs used for improving glycemic control in patients with type 2 diabetes mellitus. Two thiazolidinediones, viz. rosiglitazone and pioglitazone are currently available in the market. These drugs are the selective agonists for the nuclear peroxisome proliferator activated receptor (PPAR) gamma, which enhance the transcription of several insulin responsive genes. TZDs stimulate the expression of GLUT-4 and lower the blood glucose level primarily by increasing insulin sensitivity in peripheral tissues. These drugs also suppress hepatic gluconeogenesis [2]. Rosiglitazone and pioglitazone were approved in 1999 by USFDA and are widely prescribed drugs for type 2 diabetes mellitus by physicians all over the world. Diabetes mellitus is a major con-
∗ Corresponding author. Tel.: +91 9810590843; fax: +91 11 26059663. E-mail address: [email protected] (K. Dubey). 2210-5220/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biomag.2010.12.001
tributing factor for the cardiovascular problems and it is estimated that 65% of deaths in patients with the diabetes are from cardiovascular diseases. Recently, the cardiovascular safety of rosiglitazone has been questioned. Rosiglitazone and other thiazolidinediones are known to provoke congestive heart failure in susceptible patients. The increase in cardiovascular events may be due to adverse effect on lipid, precipitation of heart failure or reduction of hemoglobin level. Rosiglitazone is not the first TZD that has been reported to have serious adverse effect. Troglitazone, the first agent in this class approved in 1997 for glycemic control was removed from the market because of serious liver toxicity in 2000. Muraglitazar, an investigational dual PPAR gamma and alpha agonist, increased cardiovascular events including myocardial infarction during phase II and phase III testing. After reporting of cardiovascular events, muraglitazar was not approved by FDA, and further development was subsequently halted by its manufacturer. TZDs such as rosiglitazone activate or suppress dozens of genes resulting in variety of complex biological effects. These agents have shown various toxic effects during developmental stage, so despite intense interest in this class of molecules none of them reached to market in 6 years since last approval. Therefore, unexpected adverse reactions are possible while administering these drugs to the patients [3–5].
66
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
Doxorubicin (DOX) is a quinine-containing anthracycline antibiotic, an important anticancer drug used in the treatment against disseminated neoplasm. Unfortunately, the clinical use of this drug is limited by cumulative dose related cardiotoxicity, which may lead to a severe and irreversible form of cardiomyopathy. The various mechanisms responsible for the development of cardiomyopathy include, inhibition of nucleic acid and protein synthesis, release of vasoactive amines, abnormalities in mitochondria, formation of free radicals, lipid peroxidation and depletion of nonprotein tissue sulfhydryl groups. Recently, it has also been reported that apoptosis (i.e. programmed cell death) of cardiomyocyte play a crucial role in both acute and chronic cardiotoxicity of DOX [6–13]. Based on these issues and concerns, the present study was designed to investigate the effect of rosiglitazone and pioglitazone on doxorubicin-induced cardiomyopathy in rats. 2. Materials and methods 2.1. Drugs and chemicals Rosiglitazone and pioglitazone were gift samples from Panacea Biotech (Punjab, India). Doxorubicin was purchased from Dabur Research Foundation (U.P. India). All other chemicals used in the experiment were of analytical grade. HPLC grade water was used for all biochemical assays. 2.2. Animals The laboratory animal protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Hamdard University, New Delhi. Male albino rats of Wistar strain, weighing 250–300 g, were procured from the Central Animal House Facility, Hamdard University, New Delhi and acclimatized under standard laboratory conditions at 25 ± 2 ◦ C, relative humidity (50 ± 15%). The animals were kept in polypropylene cages under standard laboratory conditions (12 h light and 12 h dark: day: night cycle) and had a free access to commercial pellet diet and water ad libitum. 2.3. Experimental schedule After acclimatization, all the animals were randomly divided into six groups of eight animals each and treated as follows: Group I (normal vehicle control) – rats received 0.5% carboxymethyl cellulose (C.M.C) in water for 14 days; Group II (DOX alone treated) – rats treated with 0.5% C.M.C for 14 days and doxorubicin (15 mg/kg i.p. single dose) was administered on 10th day; Group III (rosiglitazone per se) – rats received rosiglitazone (5 mg/kg p.o.) for 14 days; Group IV (rosiglitazone + doxorubicin) – rats treated with rosiglitazone (5 mg/kg p.o.) for 14 days and doxorubicin (15 mg/kg i.p. single dose) was administered on 10th day; Group V (pioglitazone per se) – rats were given pioglitazone (10 mg/kg p.o.) for 14 days; Group VI (pioglitazone + doxorubicin) – rats treated with pioglitazone (10 mg/kg p.o.) for 14 days and doxorubicin (15 mg/kg i.p. single dose) was injected on 10th day. After completion of the 14 days treatment, blood samples were collected from retro-orbital plexus in ice-cold containers without anticoagulant; the serum was separated by centrifugation (4000 rpm, 10 min) and transferred to eppendorf tubes for the estimation of serum lactate dehydrogenase (LDH) and lipid profile.
measurement of LDH was based on the principle that LDH catalyzes the conversion of pyruvate to lactate; reduced nicotinamide adenine dinucleotide (NADH) is oxidized to nicotinamide adenine dinucleotide (NAD) in the process. The rate of decrease in NADH is directly proportional to the LDH activity [14]. 2.4.2. Estimation of lipid profile Serum total cholesterol (TC) and triglycerides (TGs) were determined by enzymatic colorimetric assay using commercial diagnostic kits supplied by Span Diagnostics, Surat, India. Highdensity lipoprotein (HDL) levels were determined by the kit purchased from Reckon Diagnostic P. Ltd, Baroda, India. The level of LDL and VLDL cholesterol were calculated as described by Friedwald equation [15]. 2.5. Preparation of heart tissue homogenates All the rats were anaesthetized and sacrificed by cervical dislocation; heart tissues were excised, washed in ice-cold saline and homogenized in 0.1 M Tris–HCl–0.001 M EDTA buffer (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 ◦ C. The supernatant was collected and used for the experiments. The protein content of the experimental samples was measured by the method of Lowary et al. using bovine serum albumin as standard [16]. 2.5.1. Biochemical assay in heart tissue homogenates Measurement of lipid peroxidation (TBARS) by determination of myocardial malondialdehyde content was performed by method of Ohkawa et al. [17]. The antioxidant enzymes namely glutathione, superoxide dismutase and catalase were determined in cardiac tissue as per standard protocol. Glutathione was estimated by the Ellman method [18]. The activity of SOD was measured by Marklund et al. [19]. Catalsae activity was measured according to the method of Clairborne [20]. 2.6. Transmission electron microscopic examination Samples were fixed in modified Karnovasky’s fluid, buffered with 0.1 M sodium phosphate buffers at pH 7.4. Fixation was done for 10–18 h at 4 ◦ C. After this, the tissues were washed in fresh buffer, and post fixed for 2 hours in 1% osmium tetraoxide in the same buffer at 4 ◦ C. After several washes in 0.1 N sodium phosphate buffers, the specimens were dehydrated in graded acetone solutions and embedded in CY212. Ultrathin sections of 60–80 nm thickness were cut using an ultracut E (Reichert Jung) ultramicrotome and the sections were stained in alcoholic urbanely acetate (10 min) and lead citrate (10 min) before examining the grids in transmission electron microscope (FEI-Margagni operated 268) Netherlands at 6080 kv [21]. 2.7. Histopathological studies The cardiac tissues were fixed in 10 % formalin solution. The specimens were processed by the standard procedure and suspended in paraffin wax. The ventricular portion was used for section preparation and five micron thick sections were stained by using hematoxylin and eosin (H. & E.). The prepared sections were examined under a light microscope.
2.4. Biochemical estimations in serum
2.8. Statistical analysis
2.4.1. Determination of serum lactate dehydrogenase (LDH) The serum LDH activity was measured by a kit using a U.V spectrophotometer (optimized standard kit Roche/Hitachi). The
Statistical analysis was carried out using Graph pad 3.0 (Graph pad software; San Diego, CA). All results are expressed as mean ± SEM. Groups of data were compared with an analysis of
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71 Table 1 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in heart weight/body weight ratio and mortality in rats. Groups
Heart weight/body weight ratio ×10−3
Mortality (%)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
2.91 ± 0.41 2.01 ± 0.71** 2.67 ± 0.10*
0 12.5 0
1.61 ± 0.90***,****
25
2.71 ± 0.20
*
1.81 ± 0.71***,****
0 12.5
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; **** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
variance followed by Bonferroni’s test. Values were considered significant at p < 0.05. 3. Results 3.1. General observations and mortality The general appearance of all animals was recorded during the entire experimental period. The animals’ fur became scruffy and developed a light yellow tinge and there were red exudates around the eyes in all the groups treated with doxorubicin alone and/or in combination with rosiglitazone and pioglitazone. The development of a grossly enlarged abdomen was the most predominant feature of the DOX alone treated group. Animals treated with doxorubicin alone and/or in combination with rosiglitazone and pioglitazone appeared to be sick, weak and lethargic and diarrhea was the most predominant feature of rosiglitazone + doxorubicin treated group. During the post treatment period (after DOX administration), 12.5% mortality was observed in the DOX and PIO + DOX treated group whereas 25% mortality was observed in ROSI + DOX treated group (Table 1). No mortality was observed in normal control group, rosiglitazone per se and pioglitazone per se treated groups. Data on heart weight/body weight ratio are given in Table 1. Heart weight/body weight ratio was significantly lowered in the rosiglitazone per se (p < 0.05), DOX (p < 0.001), ROSI + DOX, and PIO + DOX (p < 0.001) treated groups as compared with control group. 3.2. Biochemical results 3.2.1. Lactate dehydrogenase (LDH) There was a significant (p < 0.001) rise in serum LDH activity in DOX, rosiglitazone per se and pioglitazone per se groups when compared with control group (Table 2). The treatment with ROSI + DOX and PIO + DOX showed a significant (p < 0.001, p < 0.05 respectively) increase in the serum LDH level as compared to DOX treated group (Table 2). 3.2.2. Thiobarbituric acid reactive substance (TBARS) TBARS levels were determined by evaluating myocardial malondialdehyde (MDA) content. TBARS levels were significantly (p < 0.001) higher in DOX treated, rosiglitazone per se and pioglitazone per se treated groups as compared to the control group (Table 2). TBARS levels were also significantly (p < 0.001, p < 0.01) higher in the ROSI + DOX and PIO + DOX treated groups when compared with the DOX treated group (Table 2).
67
Table 2 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in serum LDH and cardiac tissue TBARS in rats. Groups
Lactate dehydrogenase (LDH IU/L)
Thiobarbituric acid reactive substance (TBARS) (nmol MDA/mg protein)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
24.85 ± 1.68 256.60 ± 5.10*** 111.45 ± 7.89***
3.98 ± 0.01 19.03 ± 0.07*** 6.02 ± 0.08***
294.10 ± 5.12******
28.82 ± 0.19******
109.15 ± 8.87***
5.41 ± 0.04***
283.55 ± 3.94****
25.12 ± 0.10*****
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; **** p < 0.05, ***** p < 0.01, ****** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
3.2.3. Serum triglyceride (TGs) There was a significant (p < 0.001) rise in serum triglyceride level in the DOX alone treated group as compared to control group. However, the levels were significantly lowered (p < 0.001) in rosiglitazone per se and pioglitazone per se treated groups when compared with control group (Table 3). The levels of TGs were also significantly reduced (p < 0.001) in ROSI +DOX and PIO + DOX treated group as compared to DOX alone treated group (Table 3). 3.2.4. Serum total cholesterol (TC) The serum total cholesterol level increased significantly (p < 0.001) in DOX, rosiglitazone per se and pioglitazone per se treated groups as compared to control group (Table 3). Total cholesterol level was also raised significantly (p < 0.001) in the ROSI + DOX and PIO + DOX treated group when compared with DOX alone treated group (Table 3). 3.2.5. High density lipoprotein (HDL-C) There was no significant difference in serum HDL-C level of DOX alone treated group compared to normal control group (Table 3). The serum HDL-C level decreased significantly (p < 0.001) in rosiglitazone per se and ROSI + DOX treated group and increased significantly (p < 0.001) in pioglitazone per se and PIO + DOX treated group when compared with control group and toxic control group respectively (Table 3). 3.2.6. Low density lipoprotein (LDL-C) The serum LDL-C level increased significantly (p < 0.001) in DOX alone treated group and rosiglitazone per se treated group, however, the value reduced significantly (p < 0.001) in pioglitazone per se treated group, as compared to control group (Table 3). The LDLC level was raised significantly (p < 0.001) in ROSI + DOX treated group however no significant difference was observed in PIO + DOX treated group when compared to DOX treated group (Table 3). 3.2.7. Very low density lipoprotein (VLDL-C) The level of serum VLDL-C increased significantly (p < 0.001) in DOX treated group and decreased significantly (p < 0.001) in rosiglitazone per se treated group (Table 3). The level of VLDL-C reduced significantly in ROSI + DOX (p < 0.05) and in PIO + DOX treated group (p < 0.001) as compared to DOX treated group (Table 3). 3.2.8. Antioxidant enzymes GSH and SOD levels were significantly (p < 0.001) lowered in the DOX, rosiglitazone per se and pioglitazone per se treated groups as compared to control group whereas CAT levels were significantly
68
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
Table 3 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in serum total cholesterol, triglycerides, HDL-C, LDL-C, and VLDL-C in rats. Groups
Triglycerides (TG’s) (mg/dl)
Total cholesterol (TC) (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
VLDL-C (mg/dl)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
45.96 ± 1.21 75.47 ± 1.62***
107.61 ± 0.78 138.08 ± 0.52***
18.775 ± 0.56 21.31 ± 1.31n.s.
82.09 ± 1.21 102.60 ± 1.30***
9.69 ± 0.54 15.28 ± 0.77***
34.28 ± 2.10***
113.45 ± 0.56***
4.732 ± 0.49***
99.65 ± 2.48***
6.05 ± 0.21***
54.35 ± 1.29*****
166.15 ± 0.92*****
12.46 ± 0.65*****
144.79 ± 1.38*****
12.42 ± 0.78****
25.23 ± 2.63***
143.41 ± 1.21***
61.25 ± 1.51***
71.11 ± 1.12***
8.18 ± 0.41n.s.
36.40 ± 1.82*****
193.26 ± 1.17*****
72.58 ± 1.02*****
105.72 ± 1.27n.s.
6.57 ± 0.29*****
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 and n.s. (non significant) when compared to normal control group, ANOVA followed by Bonferroni test; compared with DOX group, ANOVA followed by Bonferroni test.
****
p < 0.05 and
*****
p < 0.001 when
Table 4 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in cardiac tissue GSH, SOD and CAT in rats. Groups
GSH (/mg protein)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
17.04 12.12 15.12 6.12 16.21 8.89
± ± ± ± ± ±
0.17 0.21*** 0.07*** 0.10***** 0.12*** 0.02**
SOD (U/mg protein) 4.99 4.15 4.66 3.06 4.71 3.10
± ± ± ± ± ±
0.19 0.21*** 0.61*** 0.81***** 0.12*** 0.21*****
CAT (nmoL/H2 O2 min/mg protein) 12.12 20.19 15.51 26.12 14.12 24.12
± ± ± ± ± ±
0.12 0.72*** 0.21*** 0.91****** 0.20*** 0.15******
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; ***** p < 0.01 and ****** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
(p < 0.001) raised in the DOX, rosiglitazone per se and pioglitazone per se treated groups when compared to the control group (Table 4). GSH and SOD levels were also significantly (p < 0.01) lowered and CAT level was also significantly (p < 0.001) raised in ROSI + DOX and PIO + DOX treated groups as compared to the DOX treated group (Table 4). 3.2.9. Transmission electron microscopy Electron microscopy of myocardium of normal control group showed (Fig. 1a) normal architecture of cardiac myofibrils, nucleus with well-preserved nuclear membrane and chromatin network. The DOX alone treated toxic control group revealed (Fig. 1b) damaged cardiac myofibrils, condensed chromatin marginates of nuclear membrane and cytoplasmic vacuolization. The rosiglitazone per se and pioglitazone per se treated groups (Fig. 1c and e) showed less condensation of chromatin network marginates and cytoplasmic vacuolization. However the ROSI + DOX and PIO + DOX treated groups showed (Fig. 1d and f) severe damage to myofibrils, much more condensed chromatin at the margin of the nuclear membrane and cytoplasmic vacuolization as compared to the DOX treated group. 3.2.10. Histopathology Light photomicrographs of cross sectional view of rat’s myocardium revealed (Fig. 2a) normal cardiac muscle structure with well preserved cytoplasm and myofibrils in control group. The DOX treated group showed (Fig. 2b) loss of myofibrils, edema and vacuolization as a sign of cardiac toxicity. The treatment of rosiglitazone per se and pioglitazone per se groups revealed (Fig. 2c and e) mild loss of myofibrils and edema as compared to the control group. ROSI + DOX and PIO + DOX treated groups showed (Fig. 2d and f) extensive loss of myofibril, vacuolization and edema as compared with the DOX treated group.
4. Discussion The present study has shown that rosiglitazone and pioglitazone, the PPAR gamma receptor agonists have aggravated doxorubicin induced cardiomyopathy in rats. Repeated administration of doxorubicin beyond a certain dose has been shown to cause cardiomyopathy in patients as well as in a variety of animal models [13]. Doxorubicin undergoes biotransformation by cytochrome P450 and flavin enzymes in the cardiac myocyte and generates quinine type free radicals that react with oxygen to generate superoxide radical. This superoxide radical damages several macromolecular cellular components and causes lipid and protein per oxidation [22]. In this study DOX produced significant (p < 0.001) cardiomyopathy as revealed by increased serum activity of clinical cardiac marker LDH, cardiac tissue TBARS and decreased activity of SOD and glutathione in cardiac tissue as compared with control group. Doxorubicin also produced hyperlipidemia [23] as evidenced by increased serum level of total cholesterol, TGs, and LDL level. Mechanism of DOX induced hyperlipidemia is thought to be related to increased hepatic lipoprotein synthesis and secretion, reduction of lipid storage [24] and decrease in lipoprotein lipase activity [25]. The present study exhibited a significant (p < 0.001) rise in the LDH and TBARS activity in the rosiglitazone and pioglitazone treated groups either alone and also in combination with doxorubicin, compared with control and DOX alone treated group respectively, indicating that rosiglitazone and pioglitazone have aggravated cardiotoxicity induced by doxorubicin. Increased TBARS associated with enhanced lipid peroxidation suggest that increased ROS formation could be one of the mechanisms through which rosiglitazone and pioglitazone aggravate doxorubicin induced cardiomyopathy [26,27].
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
69
Fig. 1. Transmission electron microscopy of myocardial cells. (a) Normal control group showing normal nucleus (N), myofibril (MF). (b) Doxorubicin treated group showing damaged nuclear membrane, condensed chromatin marginates at nuclear membrane. (c and e) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) alone treated group showing slightly condensed chromatin marginate. (d and f) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) along with doxorubicin treated group showing extensive damaged nucleus, condensed chromatin marginates at nuclear membrane.
A high level of cholesterol in the blood stream is one of the major contributing factors for cardiac disease; therefore lipid profile analysis is a valuable marker for the diagnosis of heart problems [28]. In the present study administration of rosiglitazone alone and also in combination with doxorubicin raised total cholesterol and LDL level
significantly (p < 0.001) as compared with the control and toxic control group respectively. However, in the same way pioglitazone administration lowered LDL level and enhanced significantly (p < 0.001) HDL level, indicating a shift towards benefit. The lipid lowering effect of pioglitazone may be the result of its direct agonist
Fig. 2. Photomicrographs of rat’s heart tissue. (a) Normal control group reveals normal cardiac myofibrils with well-preserved cytoplasm. (b) Doxorubicin treated group showing damaged myofibrils and edema. (c and e) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) per se treated group showing slightly damaged myofibrils and edema. (d and f) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) along with doxorubicin treated group reveals extensive damage to myofibrils, vacuolization and edema (H and E, ×10).
70
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
action on PPAR alpha-receptors [29]. However, the effect of pioglitazone on cholesterol profile which shows a shift towards benefit alone cannot determine its cardio protective potential because pioglitazone alone and in combination with doxorubicin, also significantly raised the level of LDH, reduced antioxidant enzymes (GSH, SOD) in cardiac tissue and damaged myofibrils as revealed in histopathology and TEM findings as compared to normal and toxic control group respectively. The amount of oxidative stress was assessed by measuring the level of endogenous antioxidant enzymes (SOD, GSH and CAT). The activity of GSH and SOD was significantly (p < 0.01, p < 0.001) reduced and of CAT was significantly (p < 0.001) raised in cardiac tissue of rosiglitazone and pioglitazone treated groups either alone and in combination with DOX as compared with control and DOX treated groups respectively. These results suggest that administration of rosiglitazone and pioglitazone reduced the antioxidant enzymes and responsible for aggravation of DOX induced cardiomyopathy. The role of oxidative stress in the development of cardiomyopathy may further be supported by the increased CAT activity in the cardiac tissue. The CAT enzyme is responsible for the decomposition of hydrogen peroxide to water and oxygen molecule, so increased CAT activity in the cardiac tissue of rats treated with DOX might be the result of induction of CAT enzyme [30,31]. Besides biochemical observations transmission electron microscopy and histopathology also revealed that rosiglitazone and pioglitazone augmented DOX induced cardiomyopathy. TEM studies had shown damaged myofibrils, condensation of chromatin network, vacuolization and cell shrinkage; the sign of cell apoptosis when rosiglitazone and pioglitazone were given either alone and in combination with doxorubicin. Similarly, histopathological findings suggested damaged myofibrils, edema and vacuolization in the rats treated with rosiglitzone and pioglitazone alone and the sign of cell damage was further exaggerated when these drugs were given along with doxorubicin. Recent studies suggest that TZDs are associated with heart failure and certain type of fractures [32]. Our study supports the finding of Nissen and Wolsky that rosiglitazone is associated with increased risk of myocardial infarction and an increased risk of death from cardiovascular causes [3]. Singh and Loke concluded that both rosiglitazone and pioglitazone increase the risk of heart failure probably through a complex gene expression in the kidney involved in fluid retention [33]. Interim analysis of RECORD trial [34], DREAM trial [35] and ADOPT trial [36] showed an increased risk of heart failure associated with rosiglitazone use which is consistent with the findings of our study. Based on this study, we conclude that both rosiglitazone and pioglitazone are cardiotoxic in this model of doxorubicin induced cardiomyopathy in rats. Therefore, clinicians are advised to remain alert for the development of cardiovascular events while using TZDs and it is also important to consider benefit of these agents in the context of other oral anti diabetic drugs. Further, more preclinical and pharmacovigilance data is needed to quantify the cardiovascular risk associated with these drugs. 5. Conclusion In conclusion, the present study demonstrates that rosiglitazone and pioglitazone aggravate doxorubicin induced cardiomyopathy in rats. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgment The study was supported by a postgraduate scholarship from the University Grants Commission (UGC), Government of India New Delhi, India. References [1] Gorgino F, Laviola L, Leonandini A. A pathophysiology of type 2 diabetes: rationale for different oral antidiabetic treatment strategies. Diabetes Res Clin Practice 2005;68:S22–9. [2] Elte JW, Blickle JF. Thiazolidinediones for the treatment of type 2 diabetes. Eur J Int Med 2007;18(1):18–25. [3] Nissen SE, Wolsky K. Effect of rosiglitazone on the risk of myocardial infraction and deaths from cardiovascular causes. New Eng J Med 2007;356(24):2457–70. [4] News. Weighing up the evidence. Nature 2007;447:512–3. [5] El-Hage. Peroxisome proliferators activated receptor (PPAR) agonist: clinical and cardiac safety considerations 2007. [Accessed: www.fda.oc/cder/present/dia2006/El-Hage cardiac safety.ppt]. [6] Coudray C, Hida H, Boucher F, et al. Modulation by selenium supplementation of lipid peroxidation induced by chronic administration in rats. Nutrition 1995;11:512–6. [7] Singal PK, Iliskovik N. Doxorubicin-induced cardiomyopathy. N Eng J Med 1998;339:900–5. [8] Sorenson K, Lewitt G, Bull C, et al. Anthracycline dose in childhood acute lymphoblastic leukemia: issues of early survival and late cardiotoxicity. J Clin Oncol 1997;15:61–8. [9] Doroshow JH. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res 1983;43:460–72. [10] Singal PK, Iliskovik N, Li T, et al. Adriamycin cardiomyopathy: pathophysiology and prevention. Fed Am Soc Exp Biol 1997;11:931–6. [11] Myers CE, McGuire WP, Liss RH, et al. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science 1977;19:165–7. [12] Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–28. [13] Singh BK, Pathan RA, Pillai KK, et al. Diclofenac sodium. A nonselective nonsteroidal anti-Inflammatory drug, aggravates doxorubicin induced cardiomyopathy in rats. J Cardiovascular Pharmacol 2010;55(2):139–44. [14] Lum G, Gambino SR. A comparision of serum versus heparinized plasma for routine chemistry test. American J Clinical Pathol 1974;62:108. [15] Friedwald WT, Levy RI, Fredrickson DS. Estimation of concentration of LDL cholesterol in plasma without use of preparative ultracentrifuge. Clinical Chem 1972;18:499. [16] Lowary OH, Rosebrough NJ, Farr AL, et al. Protein measuremet with the folin phenol measurement. J Biol Chem 1951;193:265–75. [17] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reactions. Anal Biochem 1979;95:359–64. [18] Sedlak J, Lindsay RH. Estimation of total, protein bound and nonprotein sulfhydryl group in tissue with Ellman’s reagent. Anal biochem 1968;25:192–205. [19] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation by pyrogallol and convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469–74. [20] Clairborne A. Assay of catalase. In: Greenwald RA, editor. Handbook of Methods of Oxygen Free Radicals Research. Boca Raton, FL:CRC Press; 1985. p. 283–284. [21] David GFX, Herbert J, Wright CDS. The ultrastructre of pineal ganglion in the ferret. J Anat 1973;115:79. [22] Gosalvez M, Van Rossuns GD, Blanco MF. Inhibition of sodium potassium acti vated adenosine 5 triphosphate and ion transport by adriamycin. Cancer Res 1979;39:257–61. [23] Bertani T, Pozzoni R, Delaini F, et al. Adriamycin-induced nephrotic syndrome in rats:sequence of pathologic events. Lab Inves 1982;46(1):16–23. [24] Bizzi A, Ceriani L, Gerundino M, et al. Adriamycin causes hyperlipidemia as a consequence of nephrotoxicity. Toxicol Lett 1983;18(3):291–300. [25] Kashyap ML, DeMendoza SG, Campbell M, et al. Lipoprotein lipase activator deficiency in very low density lipoproteins in rat nephritic syndrome. Experientia 1978;34(8):1044–5. [26] Sevanian A, Hochstein P. Mechanisms and consequences of lipid peroxidation in biological systems. Ann Rev Nutr 1985;5:365–90. [27] Hickey EJ, Raje RR, Reid VE, et al. Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death. Free Radic Biol Med 2001;31:139–52. [28] Ito H, Nakano I, Kinoshita M, et al. Pioglitazone, a peroxisome proliferator-activated receptor- gamma agonist, attenuates myocardial ischemia/reperfusion injury in a rat model. Lab Inves 2003;83:1715–21. [29] Goldbourt U, Yaari S, Medalie JH. Isolated Low HDL Cholesterol as a risk factor for coronary heart disease mortality: a 21-year follow-up of 8000 man. Arterioscl Thromb Vasc Biol 1997;17:107–13. [30] Timao L, Singal PK. Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation 2000;102:2105–10. [31] Bassenge E, Sommer O, Schwenmmer M, et al. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. Am J Physiol Heart Circ Physiol 2000;279:H2431–8.
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71 [32] Yki-Jarvinen H, Thiazolidinedione. New Eng J Med 2004;351:1106–8. [33] Singh S, Loke YK. Thiazolidinediones and heart failure: a teleo analysis. Diabetes Care 2007;30:2148–53. [34] Home PD. Rosiglitazone evaluated for cardiovascular outcomes- An interim analysis. New Eng J Med 2007;367:28–38.
71
[35] Gerstein HC, Yusuf S, Holman RR, et al. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance on impaired fasting glucose: A randomized controlled trial (DREAM). Lancet 2006;6737(66):69420–8. [36] Kahn S, Haffiner S, Heise M, et al. Glycemic durability of rosiglitazone metformin or glyburide monotherapy. New Eng J Med 2006;355(23):2427–43.
Original article
Rosiglitazone and pioglitazone aggravate doxorubicin-induced cardiomyopathy in Wistar rats Priyadarshan Saraogi , Krishna Kolappa Pillai , Bhulan Kumar Singh , Kiran Dubey ∗ Department of pharmacology, faculty of pharmacy, Jamia Hamdard (Hamdard University), 110062 New Delhi, India
a r t i c l e
i n f o
Article history: Received 18 September 2010 Accepted 6 December 2010 Keywords: Rosiglitazone Cardiomyopathy Thiazolidinediones
a b s t r a c t Recently the cardiovascular safety of thiazolidinediones (TZDs) has been questioned. This study was designed to assess the effect of rosiglitazone (ROSI 5 mg/kg p.o.) and pioglitazone (PIO 10 mg/kg p.o.) on doxorubicin (DOX 15 mg/kg i.p. single dose) induced cardiomyopathy in Wistar rats. DOX (15 mg/kg i.p. single dose) induced cardiomyopathy was evidenced by a significant (p < 0.001) increase in serum lactate dehydrogenase (LDH), cholesterol profile, thiobarbituric acid reactive substance (TBARS), catalase (CAT) levels and a significant (p < 0.001) decrease in glutathione and superoxide dismutase levels followed by histopathological and transmission electron microscopic changes. Rosiglitazone (5 mg/kg p.o.) and pioglitazone (10 mg/kg p.o.) were administered for 14 days and doxorubicin (15 mg/kg i.p. single dose) was injected on the 10th day of drug treatment. The biochemical estimations revealed that treatment with both these drugs significantly (p < 0.001) increased the serum LDH, cardiac TBARS and catalase levels, while the antioxidant enzyme levels of glutathione and superoxide dismutase were reduced. The administration of rosiglitazone significantly (p < 0.001) reduced the level of serum high density lipoprotein (HDL) and raised the level of low density lipoprotein (LDL) while pioglitazone treatment had shown entirely opposite trend; significantly (p < 0.001) increased HDL level and lowered the LDL level. The transmission electron microscopy and histopathology of rat’s cardiac tissue, however, revealed that treatment with both these drugs caused extensive damage to the myocardium as evidenced by condensed chromatin marginates, loss of myofibrils and vacuolization and aggravated doxorubicin-induced cardiomyopathy. © 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a group of syndrome characterized by hyperglycemia, altered metabolism of lipids, carbohydrate, proteins and increased risk of complications from cardiovascular diseases [1]. Thiazolidinediones (TZDs) is a novel class of oral antidiabetic drugs used for improving glycemic control in patients with type 2 diabetes mellitus. Two thiazolidinediones, viz. rosiglitazone and pioglitazone are currently available in the market. These drugs are the selective agonists for the nuclear peroxisome proliferator activated receptor (PPAR) gamma, which enhance the transcription of several insulin responsive genes. TZDs stimulate the expression of GLUT-4 and lower the blood glucose level primarily by increasing insulin sensitivity in peripheral tissues. These drugs also suppress hepatic gluconeogenesis [2]. Rosiglitazone and pioglitazone were approved in 1999 by USFDA and are widely prescribed drugs for type 2 diabetes mellitus by physicians all over the world. Diabetes mellitus is a major con-
∗ Corresponding author. Tel.: +91 9810590843; fax: +91 11 26059663. E-mail address: [email protected] (K. Dubey). 2210-5220/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biomag.2010.12.001
tributing factor for the cardiovascular problems and it is estimated that 65% of deaths in patients with the diabetes are from cardiovascular diseases. Recently, the cardiovascular safety of rosiglitazone has been questioned. Rosiglitazone and other thiazolidinediones are known to provoke congestive heart failure in susceptible patients. The increase in cardiovascular events may be due to adverse effect on lipid, precipitation of heart failure or reduction of hemoglobin level. Rosiglitazone is not the first TZD that has been reported to have serious adverse effect. Troglitazone, the first agent in this class approved in 1997 for glycemic control was removed from the market because of serious liver toxicity in 2000. Muraglitazar, an investigational dual PPAR gamma and alpha agonist, increased cardiovascular events including myocardial infarction during phase II and phase III testing. After reporting of cardiovascular events, muraglitazar was not approved by FDA, and further development was subsequently halted by its manufacturer. TZDs such as rosiglitazone activate or suppress dozens of genes resulting in variety of complex biological effects. These agents have shown various toxic effects during developmental stage, so despite intense interest in this class of molecules none of them reached to market in 6 years since last approval. Therefore, unexpected adverse reactions are possible while administering these drugs to the patients [3–5].
66
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
Doxorubicin (DOX) is a quinine-containing anthracycline antibiotic, an important anticancer drug used in the treatment against disseminated neoplasm. Unfortunately, the clinical use of this drug is limited by cumulative dose related cardiotoxicity, which may lead to a severe and irreversible form of cardiomyopathy. The various mechanisms responsible for the development of cardiomyopathy include, inhibition of nucleic acid and protein synthesis, release of vasoactive amines, abnormalities in mitochondria, formation of free radicals, lipid peroxidation and depletion of nonprotein tissue sulfhydryl groups. Recently, it has also been reported that apoptosis (i.e. programmed cell death) of cardiomyocyte play a crucial role in both acute and chronic cardiotoxicity of DOX [6–13]. Based on these issues and concerns, the present study was designed to investigate the effect of rosiglitazone and pioglitazone on doxorubicin-induced cardiomyopathy in rats. 2. Materials and methods 2.1. Drugs and chemicals Rosiglitazone and pioglitazone were gift samples from Panacea Biotech (Punjab, India). Doxorubicin was purchased from Dabur Research Foundation (U.P. India). All other chemicals used in the experiment were of analytical grade. HPLC grade water was used for all biochemical assays. 2.2. Animals The laboratory animal protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Hamdard University, New Delhi. Male albino rats of Wistar strain, weighing 250–300 g, were procured from the Central Animal House Facility, Hamdard University, New Delhi and acclimatized under standard laboratory conditions at 25 ± 2 ◦ C, relative humidity (50 ± 15%). The animals were kept in polypropylene cages under standard laboratory conditions (12 h light and 12 h dark: day: night cycle) and had a free access to commercial pellet diet and water ad libitum. 2.3. Experimental schedule After acclimatization, all the animals were randomly divided into six groups of eight animals each and treated as follows: Group I (normal vehicle control) – rats received 0.5% carboxymethyl cellulose (C.M.C) in water for 14 days; Group II (DOX alone treated) – rats treated with 0.5% C.M.C for 14 days and doxorubicin (15 mg/kg i.p. single dose) was administered on 10th day; Group III (rosiglitazone per se) – rats received rosiglitazone (5 mg/kg p.o.) for 14 days; Group IV (rosiglitazone + doxorubicin) – rats treated with rosiglitazone (5 mg/kg p.o.) for 14 days and doxorubicin (15 mg/kg i.p. single dose) was administered on 10th day; Group V (pioglitazone per se) – rats were given pioglitazone (10 mg/kg p.o.) for 14 days; Group VI (pioglitazone + doxorubicin) – rats treated with pioglitazone (10 mg/kg p.o.) for 14 days and doxorubicin (15 mg/kg i.p. single dose) was injected on 10th day. After completion of the 14 days treatment, blood samples were collected from retro-orbital plexus in ice-cold containers without anticoagulant; the serum was separated by centrifugation (4000 rpm, 10 min) and transferred to eppendorf tubes for the estimation of serum lactate dehydrogenase (LDH) and lipid profile.
measurement of LDH was based on the principle that LDH catalyzes the conversion of pyruvate to lactate; reduced nicotinamide adenine dinucleotide (NADH) is oxidized to nicotinamide adenine dinucleotide (NAD) in the process. The rate of decrease in NADH is directly proportional to the LDH activity [14]. 2.4.2. Estimation of lipid profile Serum total cholesterol (TC) and triglycerides (TGs) were determined by enzymatic colorimetric assay using commercial diagnostic kits supplied by Span Diagnostics, Surat, India. Highdensity lipoprotein (HDL) levels were determined by the kit purchased from Reckon Diagnostic P. Ltd, Baroda, India. The level of LDL and VLDL cholesterol were calculated as described by Friedwald equation [15]. 2.5. Preparation of heart tissue homogenates All the rats were anaesthetized and sacrificed by cervical dislocation; heart tissues were excised, washed in ice-cold saline and homogenized in 0.1 M Tris–HCl–0.001 M EDTA buffer (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 ◦ C. The supernatant was collected and used for the experiments. The protein content of the experimental samples was measured by the method of Lowary et al. using bovine serum albumin as standard [16]. 2.5.1. Biochemical assay in heart tissue homogenates Measurement of lipid peroxidation (TBARS) by determination of myocardial malondialdehyde content was performed by method of Ohkawa et al. [17]. The antioxidant enzymes namely glutathione, superoxide dismutase and catalase were determined in cardiac tissue as per standard protocol. Glutathione was estimated by the Ellman method [18]. The activity of SOD was measured by Marklund et al. [19]. Catalsae activity was measured according to the method of Clairborne [20]. 2.6. Transmission electron microscopic examination Samples were fixed in modified Karnovasky’s fluid, buffered with 0.1 M sodium phosphate buffers at pH 7.4. Fixation was done for 10–18 h at 4 ◦ C. After this, the tissues were washed in fresh buffer, and post fixed for 2 hours in 1% osmium tetraoxide in the same buffer at 4 ◦ C. After several washes in 0.1 N sodium phosphate buffers, the specimens were dehydrated in graded acetone solutions and embedded in CY212. Ultrathin sections of 60–80 nm thickness were cut using an ultracut E (Reichert Jung) ultramicrotome and the sections were stained in alcoholic urbanely acetate (10 min) and lead citrate (10 min) before examining the grids in transmission electron microscope (FEI-Margagni operated 268) Netherlands at 6080 kv [21]. 2.7. Histopathological studies The cardiac tissues were fixed in 10 % formalin solution. The specimens were processed by the standard procedure and suspended in paraffin wax. The ventricular portion was used for section preparation and five micron thick sections were stained by using hematoxylin and eosin (H. & E.). The prepared sections were examined under a light microscope.
2.4. Biochemical estimations in serum
2.8. Statistical analysis
2.4.1. Determination of serum lactate dehydrogenase (LDH) The serum LDH activity was measured by a kit using a U.V spectrophotometer (optimized standard kit Roche/Hitachi). The
Statistical analysis was carried out using Graph pad 3.0 (Graph pad software; San Diego, CA). All results are expressed as mean ± SEM. Groups of data were compared with an analysis of
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71 Table 1 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in heart weight/body weight ratio and mortality in rats. Groups
Heart weight/body weight ratio ×10−3
Mortality (%)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
2.91 ± 0.41 2.01 ± 0.71** 2.67 ± 0.10*
0 12.5 0
1.61 ± 0.90***,****
25
2.71 ± 0.20
*
1.81 ± 0.71***,****
0 12.5
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; **** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
variance followed by Bonferroni’s test. Values were considered significant at p < 0.05. 3. Results 3.1. General observations and mortality The general appearance of all animals was recorded during the entire experimental period. The animals’ fur became scruffy and developed a light yellow tinge and there were red exudates around the eyes in all the groups treated with doxorubicin alone and/or in combination with rosiglitazone and pioglitazone. The development of a grossly enlarged abdomen was the most predominant feature of the DOX alone treated group. Animals treated with doxorubicin alone and/or in combination with rosiglitazone and pioglitazone appeared to be sick, weak and lethargic and diarrhea was the most predominant feature of rosiglitazone + doxorubicin treated group. During the post treatment period (after DOX administration), 12.5% mortality was observed in the DOX and PIO + DOX treated group whereas 25% mortality was observed in ROSI + DOX treated group (Table 1). No mortality was observed in normal control group, rosiglitazone per se and pioglitazone per se treated groups. Data on heart weight/body weight ratio are given in Table 1. Heart weight/body weight ratio was significantly lowered in the rosiglitazone per se (p < 0.05), DOX (p < 0.001), ROSI + DOX, and PIO + DOX (p < 0.001) treated groups as compared with control group. 3.2. Biochemical results 3.2.1. Lactate dehydrogenase (LDH) There was a significant (p < 0.001) rise in serum LDH activity in DOX, rosiglitazone per se and pioglitazone per se groups when compared with control group (Table 2). The treatment with ROSI + DOX and PIO + DOX showed a significant (p < 0.001, p < 0.05 respectively) increase in the serum LDH level as compared to DOX treated group (Table 2). 3.2.2. Thiobarbituric acid reactive substance (TBARS) TBARS levels were determined by evaluating myocardial malondialdehyde (MDA) content. TBARS levels were significantly (p < 0.001) higher in DOX treated, rosiglitazone per se and pioglitazone per se treated groups as compared to the control group (Table 2). TBARS levels were also significantly (p < 0.001, p < 0.01) higher in the ROSI + DOX and PIO + DOX treated groups when compared with the DOX treated group (Table 2).
67
Table 2 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in serum LDH and cardiac tissue TBARS in rats. Groups
Lactate dehydrogenase (LDH IU/L)
Thiobarbituric acid reactive substance (TBARS) (nmol MDA/mg protein)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
24.85 ± 1.68 256.60 ± 5.10*** 111.45 ± 7.89***
3.98 ± 0.01 19.03 ± 0.07*** 6.02 ± 0.08***
294.10 ± 5.12******
28.82 ± 0.19******
109.15 ± 8.87***
5.41 ± 0.04***
283.55 ± 3.94****
25.12 ± 0.10*****
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; **** p < 0.05, ***** p < 0.01, ****** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
3.2.3. Serum triglyceride (TGs) There was a significant (p < 0.001) rise in serum triglyceride level in the DOX alone treated group as compared to control group. However, the levels were significantly lowered (p < 0.001) in rosiglitazone per se and pioglitazone per se treated groups when compared with control group (Table 3). The levels of TGs were also significantly reduced (p < 0.001) in ROSI +DOX and PIO + DOX treated group as compared to DOX alone treated group (Table 3). 3.2.4. Serum total cholesterol (TC) The serum total cholesterol level increased significantly (p < 0.001) in DOX, rosiglitazone per se and pioglitazone per se treated groups as compared to control group (Table 3). Total cholesterol level was also raised significantly (p < 0.001) in the ROSI + DOX and PIO + DOX treated group when compared with DOX alone treated group (Table 3). 3.2.5. High density lipoprotein (HDL-C) There was no significant difference in serum HDL-C level of DOX alone treated group compared to normal control group (Table 3). The serum HDL-C level decreased significantly (p < 0.001) in rosiglitazone per se and ROSI + DOX treated group and increased significantly (p < 0.001) in pioglitazone per se and PIO + DOX treated group when compared with control group and toxic control group respectively (Table 3). 3.2.6. Low density lipoprotein (LDL-C) The serum LDL-C level increased significantly (p < 0.001) in DOX alone treated group and rosiglitazone per se treated group, however, the value reduced significantly (p < 0.001) in pioglitazone per se treated group, as compared to control group (Table 3). The LDLC level was raised significantly (p < 0.001) in ROSI + DOX treated group however no significant difference was observed in PIO + DOX treated group when compared to DOX treated group (Table 3). 3.2.7. Very low density lipoprotein (VLDL-C) The level of serum VLDL-C increased significantly (p < 0.001) in DOX treated group and decreased significantly (p < 0.001) in rosiglitazone per se treated group (Table 3). The level of VLDL-C reduced significantly in ROSI + DOX (p < 0.05) and in PIO + DOX treated group (p < 0.001) as compared to DOX treated group (Table 3). 3.2.8. Antioxidant enzymes GSH and SOD levels were significantly (p < 0.001) lowered in the DOX, rosiglitazone per se and pioglitazone per se treated groups as compared to control group whereas CAT levels were significantly
68
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
Table 3 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in serum total cholesterol, triglycerides, HDL-C, LDL-C, and VLDL-C in rats. Groups
Triglycerides (TG’s) (mg/dl)
Total cholesterol (TC) (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
VLDL-C (mg/dl)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
45.96 ± 1.21 75.47 ± 1.62***
107.61 ± 0.78 138.08 ± 0.52***
18.775 ± 0.56 21.31 ± 1.31n.s.
82.09 ± 1.21 102.60 ± 1.30***
9.69 ± 0.54 15.28 ± 0.77***
34.28 ± 2.10***
113.45 ± 0.56***
4.732 ± 0.49***
99.65 ± 2.48***
6.05 ± 0.21***
54.35 ± 1.29*****
166.15 ± 0.92*****
12.46 ± 0.65*****
144.79 ± 1.38*****
12.42 ± 0.78****
25.23 ± 2.63***
143.41 ± 1.21***
61.25 ± 1.51***
71.11 ± 1.12***
8.18 ± 0.41n.s.
36.40 ± 1.82*****
193.26 ± 1.17*****
72.58 ± 1.02*****
105.72 ± 1.27n.s.
6.57 ± 0.29*****
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 and n.s. (non significant) when compared to normal control group, ANOVA followed by Bonferroni test; compared with DOX group, ANOVA followed by Bonferroni test.
****
p < 0.05 and
*****
p < 0.001 when
Table 4 Effect of rosiglitazone and pioglitazone on doxorubicin-induced changes in cardiac tissue GSH, SOD and CAT in rats. Groups
GSH (/mg protein)
Normal control (0.5% CMC p.o.) Doxorubicin (DOX 15 mg/kg i.p.) Rosiglitazone per se (ROSI 5 mg/kg p.o.) Rosiglitazone + doxorubicin (ROSI 5 mg/kg p.o. + DOX 15 mg/kg i.p.) Pioglitazone per se (PIO 10 mg/kg p.o.) Pioglitazone + doxorubicin (PIO 10 mg/kg p.o. + DOX 15 mg/kg i.p.)
17.04 12.12 15.12 6.12 16.21 8.89
± ± ± ± ± ±
0.17 0.21*** 0.07*** 0.10***** 0.12*** 0.02**
SOD (U/mg protein) 4.99 4.15 4.66 3.06 4.71 3.10
± ± ± ± ± ±
0.19 0.21*** 0.61*** 0.81***** 0.12*** 0.21*****
CAT (nmoL/H2 O2 min/mg protein) 12.12 20.19 15.51 26.12 14.12 24.12
± ± ± ± ± ±
0.12 0.72*** 0.21*** 0.91****** 0.20*** 0.15******
All values are expressed as mean ± SEM. n = 8 rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to normal control group, ANOVA followed by Bonferroni test; ***** p < 0.01 and ****** p < 0.001 when compared with DOX group, ANOVA followed by Bonferroni test.
(p < 0.001) raised in the DOX, rosiglitazone per se and pioglitazone per se treated groups when compared to the control group (Table 4). GSH and SOD levels were also significantly (p < 0.01) lowered and CAT level was also significantly (p < 0.001) raised in ROSI + DOX and PIO + DOX treated groups as compared to the DOX treated group (Table 4). 3.2.9. Transmission electron microscopy Electron microscopy of myocardium of normal control group showed (Fig. 1a) normal architecture of cardiac myofibrils, nucleus with well-preserved nuclear membrane and chromatin network. The DOX alone treated toxic control group revealed (Fig. 1b) damaged cardiac myofibrils, condensed chromatin marginates of nuclear membrane and cytoplasmic vacuolization. The rosiglitazone per se and pioglitazone per se treated groups (Fig. 1c and e) showed less condensation of chromatin network marginates and cytoplasmic vacuolization. However the ROSI + DOX and PIO + DOX treated groups showed (Fig. 1d and f) severe damage to myofibrils, much more condensed chromatin at the margin of the nuclear membrane and cytoplasmic vacuolization as compared to the DOX treated group. 3.2.10. Histopathology Light photomicrographs of cross sectional view of rat’s myocardium revealed (Fig. 2a) normal cardiac muscle structure with well preserved cytoplasm and myofibrils in control group. The DOX treated group showed (Fig. 2b) loss of myofibrils, edema and vacuolization as a sign of cardiac toxicity. The treatment of rosiglitazone per se and pioglitazone per se groups revealed (Fig. 2c and e) mild loss of myofibrils and edema as compared to the control group. ROSI + DOX and PIO + DOX treated groups showed (Fig. 2d and f) extensive loss of myofibril, vacuolization and edema as compared with the DOX treated group.
4. Discussion The present study has shown that rosiglitazone and pioglitazone, the PPAR gamma receptor agonists have aggravated doxorubicin induced cardiomyopathy in rats. Repeated administration of doxorubicin beyond a certain dose has been shown to cause cardiomyopathy in patients as well as in a variety of animal models [13]. Doxorubicin undergoes biotransformation by cytochrome P450 and flavin enzymes in the cardiac myocyte and generates quinine type free radicals that react with oxygen to generate superoxide radical. This superoxide radical damages several macromolecular cellular components and causes lipid and protein per oxidation [22]. In this study DOX produced significant (p < 0.001) cardiomyopathy as revealed by increased serum activity of clinical cardiac marker LDH, cardiac tissue TBARS and decreased activity of SOD and glutathione in cardiac tissue as compared with control group. Doxorubicin also produced hyperlipidemia [23] as evidenced by increased serum level of total cholesterol, TGs, and LDL level. Mechanism of DOX induced hyperlipidemia is thought to be related to increased hepatic lipoprotein synthesis and secretion, reduction of lipid storage [24] and decrease in lipoprotein lipase activity [25]. The present study exhibited a significant (p < 0.001) rise in the LDH and TBARS activity in the rosiglitazone and pioglitazone treated groups either alone and also in combination with doxorubicin, compared with control and DOX alone treated group respectively, indicating that rosiglitazone and pioglitazone have aggravated cardiotoxicity induced by doxorubicin. Increased TBARS associated with enhanced lipid peroxidation suggest that increased ROS formation could be one of the mechanisms through which rosiglitazone and pioglitazone aggravate doxorubicin induced cardiomyopathy [26,27].
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
69
Fig. 1. Transmission electron microscopy of myocardial cells. (a) Normal control group showing normal nucleus (N), myofibril (MF). (b) Doxorubicin treated group showing damaged nuclear membrane, condensed chromatin marginates at nuclear membrane. (c and e) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) alone treated group showing slightly condensed chromatin marginate. (d and f) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) along with doxorubicin treated group showing extensive damaged nucleus, condensed chromatin marginates at nuclear membrane.
A high level of cholesterol in the blood stream is one of the major contributing factors for cardiac disease; therefore lipid profile analysis is a valuable marker for the diagnosis of heart problems [28]. In the present study administration of rosiglitazone alone and also in combination with doxorubicin raised total cholesterol and LDL level
significantly (p < 0.001) as compared with the control and toxic control group respectively. However, in the same way pioglitazone administration lowered LDL level and enhanced significantly (p < 0.001) HDL level, indicating a shift towards benefit. The lipid lowering effect of pioglitazone may be the result of its direct agonist
Fig. 2. Photomicrographs of rat’s heart tissue. (a) Normal control group reveals normal cardiac myofibrils with well-preserved cytoplasm. (b) Doxorubicin treated group showing damaged myofibrils and edema. (c and e) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) per se treated group showing slightly damaged myofibrils and edema. (d and f) Rosiglitazone (5 mg/kg) and pioglitazone (10 mg/kg) along with doxorubicin treated group reveals extensive damage to myofibrils, vacuolization and edema (H and E, ×10).
70
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71
action on PPAR alpha-receptors [29]. However, the effect of pioglitazone on cholesterol profile which shows a shift towards benefit alone cannot determine its cardio protective potential because pioglitazone alone and in combination with doxorubicin, also significantly raised the level of LDH, reduced antioxidant enzymes (GSH, SOD) in cardiac tissue and damaged myofibrils as revealed in histopathology and TEM findings as compared to normal and toxic control group respectively. The amount of oxidative stress was assessed by measuring the level of endogenous antioxidant enzymes (SOD, GSH and CAT). The activity of GSH and SOD was significantly (p < 0.01, p < 0.001) reduced and of CAT was significantly (p < 0.001) raised in cardiac tissue of rosiglitazone and pioglitazone treated groups either alone and in combination with DOX as compared with control and DOX treated groups respectively. These results suggest that administration of rosiglitazone and pioglitazone reduced the antioxidant enzymes and responsible for aggravation of DOX induced cardiomyopathy. The role of oxidative stress in the development of cardiomyopathy may further be supported by the increased CAT activity in the cardiac tissue. The CAT enzyme is responsible for the decomposition of hydrogen peroxide to water and oxygen molecule, so increased CAT activity in the cardiac tissue of rats treated with DOX might be the result of induction of CAT enzyme [30,31]. Besides biochemical observations transmission electron microscopy and histopathology also revealed that rosiglitazone and pioglitazone augmented DOX induced cardiomyopathy. TEM studies had shown damaged myofibrils, condensation of chromatin network, vacuolization and cell shrinkage; the sign of cell apoptosis when rosiglitazone and pioglitazone were given either alone and in combination with doxorubicin. Similarly, histopathological findings suggested damaged myofibrils, edema and vacuolization in the rats treated with rosiglitzone and pioglitazone alone and the sign of cell damage was further exaggerated when these drugs were given along with doxorubicin. Recent studies suggest that TZDs are associated with heart failure and certain type of fractures [32]. Our study supports the finding of Nissen and Wolsky that rosiglitazone is associated with increased risk of myocardial infarction and an increased risk of death from cardiovascular causes [3]. Singh and Loke concluded that both rosiglitazone and pioglitazone increase the risk of heart failure probably through a complex gene expression in the kidney involved in fluid retention [33]. Interim analysis of RECORD trial [34], DREAM trial [35] and ADOPT trial [36] showed an increased risk of heart failure associated with rosiglitazone use which is consistent with the findings of our study. Based on this study, we conclude that both rosiglitazone and pioglitazone are cardiotoxic in this model of doxorubicin induced cardiomyopathy in rats. Therefore, clinicians are advised to remain alert for the development of cardiovascular events while using TZDs and it is also important to consider benefit of these agents in the context of other oral anti diabetic drugs. Further, more preclinical and pharmacovigilance data is needed to quantify the cardiovascular risk associated with these drugs. 5. Conclusion In conclusion, the present study demonstrates that rosiglitazone and pioglitazone aggravate doxorubicin induced cardiomyopathy in rats. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgment The study was supported by a postgraduate scholarship from the University Grants Commission (UGC), Government of India New Delhi, India. References [1] Gorgino F, Laviola L, Leonandini A. A pathophysiology of type 2 diabetes: rationale for different oral antidiabetic treatment strategies. Diabetes Res Clin Practice 2005;68:S22–9. [2] Elte JW, Blickle JF. Thiazolidinediones for the treatment of type 2 diabetes. Eur J Int Med 2007;18(1):18–25. [3] Nissen SE, Wolsky K. Effect of rosiglitazone on the risk of myocardial infraction and deaths from cardiovascular causes. New Eng J Med 2007;356(24):2457–70. [4] News. Weighing up the evidence. Nature 2007;447:512–3. [5] El-Hage. Peroxisome proliferators activated receptor (PPAR) agonist: clinical and cardiac safety considerations 2007. [Accessed: www.fda.oc/cder/present/dia2006/El-Hage cardiac safety.ppt]. [6] Coudray C, Hida H, Boucher F, et al. Modulation by selenium supplementation of lipid peroxidation induced by chronic administration in rats. Nutrition 1995;11:512–6. [7] Singal PK, Iliskovik N. Doxorubicin-induced cardiomyopathy. N Eng J Med 1998;339:900–5. [8] Sorenson K, Lewitt G, Bull C, et al. Anthracycline dose in childhood acute lymphoblastic leukemia: issues of early survival and late cardiotoxicity. J Clin Oncol 1997;15:61–8. [9] Doroshow JH. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res 1983;43:460–72. [10] Singal PK, Iliskovik N, Li T, et al. Adriamycin cardiomyopathy: pathophysiology and prevention. Fed Am Soc Exp Biol 1997;11:931–6. [11] Myers CE, McGuire WP, Liss RH, et al. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science 1977;19:165–7. [12] Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–28. [13] Singh BK, Pathan RA, Pillai KK, et al. Diclofenac sodium. A nonselective nonsteroidal anti-Inflammatory drug, aggravates doxorubicin induced cardiomyopathy in rats. J Cardiovascular Pharmacol 2010;55(2):139–44. [14] Lum G, Gambino SR. A comparision of serum versus heparinized plasma for routine chemistry test. American J Clinical Pathol 1974;62:108. [15] Friedwald WT, Levy RI, Fredrickson DS. Estimation of concentration of LDL cholesterol in plasma without use of preparative ultracentrifuge. Clinical Chem 1972;18:499. [16] Lowary OH, Rosebrough NJ, Farr AL, et al. Protein measuremet with the folin phenol measurement. J Biol Chem 1951;193:265–75. [17] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reactions. Anal Biochem 1979;95:359–64. [18] Sedlak J, Lindsay RH. Estimation of total, protein bound and nonprotein sulfhydryl group in tissue with Ellman’s reagent. Anal biochem 1968;25:192–205. [19] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation by pyrogallol and convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469–74. [20] Clairborne A. Assay of catalase. In: Greenwald RA, editor. Handbook of Methods of Oxygen Free Radicals Research. Boca Raton, FL:CRC Press; 1985. p. 283–284. [21] David GFX, Herbert J, Wright CDS. The ultrastructre of pineal ganglion in the ferret. J Anat 1973;115:79. [22] Gosalvez M, Van Rossuns GD, Blanco MF. Inhibition of sodium potassium acti vated adenosine 5 triphosphate and ion transport by adriamycin. Cancer Res 1979;39:257–61. [23] Bertani T, Pozzoni R, Delaini F, et al. Adriamycin-induced nephrotic syndrome in rats:sequence of pathologic events. Lab Inves 1982;46(1):16–23. [24] Bizzi A, Ceriani L, Gerundino M, et al. Adriamycin causes hyperlipidemia as a consequence of nephrotoxicity. Toxicol Lett 1983;18(3):291–300. [25] Kashyap ML, DeMendoza SG, Campbell M, et al. Lipoprotein lipase activator deficiency in very low density lipoproteins in rat nephritic syndrome. Experientia 1978;34(8):1044–5. [26] Sevanian A, Hochstein P. Mechanisms and consequences of lipid peroxidation in biological systems. Ann Rev Nutr 1985;5:365–90. [27] Hickey EJ, Raje RR, Reid VE, et al. Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death. Free Radic Biol Med 2001;31:139–52. [28] Ito H, Nakano I, Kinoshita M, et al. Pioglitazone, a peroxisome proliferator-activated receptor- gamma agonist, attenuates myocardial ischemia/reperfusion injury in a rat model. Lab Inves 2003;83:1715–21. [29] Goldbourt U, Yaari S, Medalie JH. Isolated Low HDL Cholesterol as a risk factor for coronary heart disease mortality: a 21-year follow-up of 8000 man. Arterioscl Thromb Vasc Biol 1997;17:107–13. [30] Timao L, Singal PK. Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation 2000;102:2105–10. [31] Bassenge E, Sommer O, Schwenmmer M, et al. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. Am J Physiol Heart Circ Physiol 2000;279:H2431–8.
P. Saraogi et al. / Biomedicine & Aging Pathology 1 (2011) 65–71 [32] Yki-Jarvinen H, Thiazolidinedione. New Eng J Med 2004;351:1106–8. [33] Singh S, Loke YK. Thiazolidinediones and heart failure: a teleo analysis. Diabetes Care 2007;30:2148–53. [34] Home PD. Rosiglitazone evaluated for cardiovascular outcomes- An interim analysis. New Eng J Med 2007;367:28–38.
71
[35] Gerstein HC, Yusuf S, Holman RR, et al. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance on impaired fasting glucose: A randomized controlled trial (DREAM). Lancet 2006;6737(66):69420–8. [36] Kahn S, Haffiner S, Heise M, et al. Glycemic durability of rosiglitazone metformin or glyburide monotherapy. New Eng J Med 2006;355(23):2427–43.