Diazoxide attenuates DOX-induced cardiotoxicity in cultured rat myocytes.

Doxorubicin (DOX)-induced cardiotoxicity is a well known clinical problem, and many investigations have been made of its possible amelioration. We have investigated whether diazoxide (DIA), an agonist at mitochondrial ATP-sensitive potassium channels (mitoKATP), could reverse DOX-induced apoptotic myocardial cell loss, in cultured rat cardiomyocytes. The role of certain proteins in this pathway was also studied. The rat cardiomyocyte cell line (H9c2) was treated with DOX, and also co-treated with DOX and DIA, for 24 h. Distribution of actin filaments, mitochondrial membrane potential, superoxide dismutase (SOD) activity, total oxidant and antioxidant status (TOS and TAS, respectively), and some protein expressions, were assessed. DOX significantly decreased SOD activity, increased ERK1/2 protein levels, and depolarised the mitochondrial membrane, while DIA co-treatment inhibited such changes. DIA co-treatment ameliorated DOX-induced cytoskeletal changes via F-actin distribution and mitoKATP structure. Co-treatment also decreased ERK1/2 and cytochrome c protein levels. Cardiomyocyte loss due to oxidative stress-mediated apoptosis is a key event in DOX-induced cytotoxicity. DIA had protective effects on DOX-induced cardiotoxicity, via mitoKATP integrity, especially with elevated SUR2A levels; but also by a cascade including SOD/AMPK/ERK1/2. Therefore, DIA may be considered a candidate agent for protecting cardiomyocytes against DOX chemotherapy.

Effects of diazoxide on streptozotocin induced ß cell damage via HSP70/HSP90/TLR4/AMPK signaling pathways.

I investigated the effects of diazoxide, a mitochondrial potassium channel opener, on streptozotocin (STZ) induced pancreatic ß cell damage via the HSP70/HSP90/TLR4/AMPK signaling pathways in vitro. I used the pancreatic ß cell line, 1.1B4, to create four groups: control, STZ treated, diazoxide treated, STZ + diazoxide treated. The STZ treated cells were exposed to 20 µM STZ for 2 h with or without 100 µM diazoxide for 24 h. Total antioxidant status (TAS), total oxidant status (TOS), cell viability and mitochondrial membrane potential (MMP) were measured. Expression of ATP-sensitive potassium channel (KATP) subunits, heat shock protein-70 (HSP70), heat shock protein-90 (HSP90), toll-like receptor 4 (TLR4), AMP-activated protein kinase (AMPK) and some apoptotic proteins were detected using western blotting. Apoptosis was assessed using TUNEL staining. STZ increased TOS and OSI in the pancreatic ß cells; however, diazoxide failed to improve oxidative stress. Also, STZ increased tunnel positive cells in the pancreatic ß cells. Diazoxide decreased the tunnel positive cells in the STZ treated ß cell. STZ decreased MMP; however, diazoxide did not normalize MMP in the STZ induced ß cells. Diazoxide increased the HSP70:HSP90 protein expression ratio. STZ decreased expression of AMPK and subunits of KATP channel and increased the expression of caspase-3 and TLR4 protein; diazoxide normalized the expression of all proteins studied. KATP channel opening by diazoxide protects pancreatic ß cells against STZ toxicity via HSP70/HSP90/TLR4/AMPK signaling.

Protective effect of propolis on myocardial ischemia/reperfusion injury in males and ovariectomized females but not in intact females.

The aim of this study is to investigate the effect of propolis, which may have estrogenic effects, on myocardial ischemia/reperfusion (mI/R) injury not only in male rats but also in intact and ovariectomized (ovx) female rats. Six groups were formed: untreated males (n = 8), treated males (n = 9), untreated intact females (n = 9), treated intact females (n = 10), untreated ovx females (n = 10), and treated ovx females (n = 8). An alcoholic extract of a single dose of propolis (200 mg/kg) was administered orally daily for 14 days. Thirty minutes of ischemia and 120 min of reperfusion were performed. Blood pressure, heart rate, arrhythmias (ventricular premature contraction [VPC], ventricular tachycardia [VT], ventricular fibrillation [VF]), and myocardial infarct size were evaluated. Total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and 17 beta-estradiol (E2) were measured. The untreated females showed more resistance to mI/R injury than the untreated males, as evidenced by lower duration, incidence, and score of arrhythmias, and smaller infarct size (p < .05). After ovx, this resistance disappeared. Propolis improved these values in treated males and treated ovx females (p < .05). Propolis increased TAS in treated males and decreased TOS in treated ovx females as well as elevated SOD in all treated groups (p < .05). Propolis decreased E2 level in treated intact females; however, it increased E2 level in treated ovx females (p < .05). The results revealed that propolis could protect the heart against mI/R injury in males and ovx females. PRACTICAL APPLICATIONS: It is known that the female heart has an increased sensitivity to myocardial ischemia/reperfusion (mI/R) injury due to estrogen deficiency and/or estrogen deprivation following menopause or surgical removal of the ovaries. Propolis has the potential to mimic estrogen under physiological and pathophysiological conditions, as well as its antioxidant property. The results indicated that propolis decreased myocardial infarct size, arrhythmia score, arrhythmia duration, and incidence in ovariectomized female rats and male rats. In addition, the present results demonstrated that an alcoholic extract of propolis as a natural product can effectively maintain the resistance of female heart to mI/R injury after estrogen deficiency.

The effect of co-administration of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rat liver.

It is possible to use plant-derived antioxidant molecules in the form of dietary supplements. However, dietary supplement-drug interaction pattern has not been well defined for most of these products. The aim of this study was to determine the effects of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rats. Streptozotocin was administered to create experimental diabetes. Resveratrol (5 mg/kg) (R), glibenclamide (5 mg/kg) (G), and berberine (10 mg/kg) (B) were administered individually or in combinations in DMSO by intraperitoneal administration route to the diabetic rats. DMSO was also given to non-diabetic control (C) and diabetic control (D) groups. Livers of rats were taken under anesthesia at the end of the treatment period (12 days). Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), aniline 4-hydroxylase (A4H), erythromycin N-demethylase (ERND), glutathione S-transferase (GST), catalase (CAT), and glutathione reductase (GR) activities were measured in microsomes and cytosols. In addition, histomorphological studies were also performed in the liver tissues. EROD activity of D+R was significantly higher than C and D+R+B. PROD activity of D+R was significantly higher than C, D, D+R+G, D+R+B, and D+R+B+ G. PROD activity of D+B was significantly higher than C and D+R+B. ERND activity of D+R was significantly higher than D+R+G and D+R+B. GST activity of D+R was significantly higher than D+R+G. CAT activity of D+B was significantly lower than C. It is clear that co-administration of resveratrol, berberine, and glibenclamide modifies some of the important xenobiotic metabolizing enzyme activities. Resveratrol and berberine have the potential to cause dietary supplement-drug interaction.

Investigation of genotoxic effects of rhododendron honey using three mammalian bioassays in vivo.

Rhododendron honey (RH) is obtained from the rhododendron plants are grown in many regions around the world, causes poisoning in humans due to the grayanotoxin (GTX) compound in its structure. It is used by the public as a therapeutic for some diseases. It was aimed to study the genotoxic and cytotoxic effects of RH in mouse bone-marrow and sperm cells by using three mammalian bioassays. 25, 50 and 75 mg kg-1 concentrations of RH given to male mice via gavage for 24 and 48 h treatment periods and its active ingredient Grayanatoxin (GTX-III) 0.01 mg kg-1 by i.p. injection. Chromosome aberrations (CA), polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE), micronucleated polychromatic erythrocytes (MNPCE) and sperm abnormalities were investigated. The results demonstrated that all the tested concentrations of RH significantly induced total abnormal cell frequency including chromosomal breaks for two time periods. In the MN assay, 75 mg kg-1 RH and 0.01 mg kg-1 GTX-III significantly increased % MNPCE and significantly reduced PCE/NCE ratios after 24 and 48 h treatments on mice demonstrating potential genotoxic and cytotoxic effect. Although there was a concentration-related increase in the percentage of total sperm abnormalities, this increase was not statistically significant compared to control. As a result, microscopic genotoxicity and cytotoxicity marker tests showed that RH and its active ingredient GTX-III have potential genotoxic and cytotoxic effect on mice bone marrow cells. It is understood that RH that is used to treat some diseases by public, should be handled carefully and used in a controlled manner.HighlightsChromosome aberration, micronucleus and sperm morphology assays are recommended as reliable biological indicators.RH and its active ingredient GTX-III have potential genotoxic and cytotoxic effect on mice bone marrow cells.Significant changes were observed upon the treatment of 75 mg kg-1 MH for MN assay.

Decreasing myocardial estrogen receptors and antioxidant activity may be responsible for increasing ischemia- and reperfusion-induced ventricular arrhythmia in older female rats.

AIMS: This study aimed to investigate the relationship between ischemia- and reperfusion-induced arrhythmia and blood serum estrogen levels, myocardial estrogen receptor levels, antioxidant enzyme activities, and the effects of the estrogen receptor blocker, fulvestrant (ICI 182 780). MAIN METHODS: A total of 102 female Sprague-Dawley rats of different ages (2-3, 6-7, 14-15, and 20-21 months) were used in this study. Myocardial ischemia was produced by ligation of the descending branch of the left anterior descending coronary artery, and reperfusion was produced by releasing this artery. An electrocardiogram (ECG) and blood pressure were recorded for 6 min of ischemia and 6 min of reperfusion. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), estrogen receptor α (ERα), and estrogen receptor ß (ERß) in myocardial tissue and 17 beta-estradiol (E2) in blood serum were measured via enzyme-linked immunosorbent assay (ELISA). The results were compared using a Mann-Whitney U test, one-way analysis of variance (ANOVA), and a student's t-test. KEY FINDINGS: It is not the changes in serum estrogen levels but the decreasing myocardial estrogen receptors and antioxidant activities that could be responsible for the occurrence of more severe arrhythmia in response to reperfusion in older female rats. SIGNIFICANCE: The death rate due to a heart attack in younger men is higher than in women. However, it equalizes after the menopausal stage in women. In this study, the reason for the increasing sudden post-menopausal death rate in women was investigated experimentally.

The role of toll-like receptors in the protective effect of melatonin against doxorubicin-induced pancreatic beta cell toxicity.

AIMS: Doxorubicin, an anticancer drug, has a toxic effect on many tissues such as heart, pancreas, liver, kidney, and testis. The aim of current study is to investigate whether melatonin would be protective in doxorubicin-induced beta (ß) cell toxicity via HMGB1/TLR2/TLR4/MAPK/NF-қB signaling pathway. MAIN METHODS: Human pancreatic ß cell (1.1B4) was used in the present study. Four experimental groups were created as control, melatonin (10 µM), doxorubicin (2 µM) and the combination of melatonin with doxorubicin. Following 24-h treatment, Mitogen-activated protein kinase (MAPKs), Toll like receptors (TLRs) including TLR2 and TLR4, pro-and anti-apoptotic protein expression levels were determined by western blotting. Total antioxidant (TAS), oxidant status (TOS) and oxidative stress index (OSI) of the cells as well as superoxide dismutase (SOD) levels were determined. Active caspase-8 activity was measured and TUNEL staining was performed to study apoptotic pathways. Mitochondrial membrane potential (MMP), some protein expressions and F-actin distribution were analyzed. KEY FINDINGS: Doxorubicin caused to depolarize MMP, resulting in enhancing apoptosis by activation of caspase-8 via MAPKs/NF-кB pathway via elevation of TOS and decreasing TAS. Also, doxorubicin destroyed F-actin distribution and elevated TLR2 and some apoptotic proteins, including Bax. However, co-treatment of melatonin with doxorubicin could reverse depolarization of MMP and inhibition of apoptosis through MAPK/NF-кB signaling by decreasing TOS and increasing TAS. The co-treatment reversed the alternations of TLR2, TLR4, MAPKs and apoptotic protein expressions induced by doxorubicin. SIGNIFICANCE: Melatonin could be a good candidate against pancreatic ß cell toxicity-induced by doxorubicin through TLR2/TLR4/MAPK/NF-кB pathways.

Inhibition of Angiotensin-II Production Increases Susceptibility to Acute Ischemia/Reperfusion Arrhythmia.

BACKGROUND Myocardial ischemia and reperfusion lead to impairment of electrolyte balance and, eventually, lethal arrhythmias. The aim of this study was to investigate the effects of pharmacological inhibition of angiotensin-II (Ang-II) production on heart tissue with ischemia-reperfusion damage, arrhythmia, and oxidative stress. MATERIAL AND METHODS Rats were divided into 4 groups: only ischemia/reperfusion (MI/R), captopril (CAP), aliskiren (AL), and CAP+AL. The drugs were given by gavage 30 min before anesthesia. Blood pressure and electrocardiography (ECG) were recorded during MI/R procedures. The heart tissue and plasma was kept so as to evaluate the total oxidant (TOS), antioxidant status (TAS), and creatine kinase-MB (CK-MB). RESULTS Creatine kinase-MB was not different among the groups. Although TAS was not affected by inhibition of Ang-II production, TOS was significantly lower in the CAP and/or AL groups than in the MI/R group. Furthermore, oxidative stress index was significantly attenuated in the CAP and/or AL groups. Captopril significantly increased the duration of VT during ischemia; however, it did not have any effect on the incidence of arrhythmias. During reperfusion periods, aliskiren and its combinations with captopril significantly reduced the incidence of other types of arrhythmias. Captopril alone had no effect on the incidence of arrhythmias, but significantly increased arrhythmias score and durations of arrhythmias during reperfusion. MAP and heart rate did not show changes in any groups during ischemic and reperfusion periods. CONCLUSIONS Angiotensin-II production appears to be associated with elevated levels of reactive oxygen species, but Ang-II inhibitions increases arrhythmia, mainly by initiating ventricular ectopic beats.

The effects of ATP-dependent potassium channel opener; pinacidil, and blocker; glibenclamide, on the ischemia induced arrhythmia in partial and complete ligation of coronary artery in rats.

OBJECTIVES: Electrical inhomogeneity between ischemic and non ischemic myocardium is the basis of arrhythmia which occurs following coronary artery occlusion. The leakage of potassium from the ischemic region to the non ischemic region is very effective in the generation of these arrhythmias. The aim of this study is to research the effect of ATP-dependent potassium (KATP) channel blocker (glibenclamide) and opener (pinacidil) on ischemia induced arrhythmia in the presence of small and large infarct sizes. MATERIALS AND METHODS: In this study Sprague-Dawley male rats of 8-9 months of age were used. Ischemia was produced by the partial ligation of left coronary artery ramus descending (PL) for smaller infarct and complete ligation of this artery (CL) for larger infarct for 30 min. The arrhythmia score which was calculated from the duration and type of arrhythmia was significantly higher in animals which had a larger infarct area than the animals which had a smaller infarct. RESULTS: Glibenclamide increased the rate of arrhythmia in animals having smaller infarct but not in animals having larger infarct. Pinacidil did not affect the occurrence of arrhythmia in either group. There was a significant difference in the infarct size and risk of infarct zone between animals which had small and large infarct sizes. The effect of glibenclamide and pinacidil on the arrhythmias differed depend on decrease of infarct size. CONCLUSION: Glibenclamide is not effective to decrease ischemia induced arrhythmia in the presence of small and pinacidil in large ischemic zone.

Effect of ATP-dependent channel modulators on ischemia-induced arrhythmia change depending on age and gender.

The number of ATP-dependent potassium channels in myocardial cells has been previously shown to change depending on gender and age. Different effects of the ATP-dependent potassium channel blocker, glybenclamide and ATP-dependent potassium channel opener, pinacidil on ischemia or reperfusion-induced arrhythmia observed in various research might depend on different ages and genders of the animals used. The aim of this study is to research the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia in animals of different ages and genders. Sprague-Dawley rats of different ages and genders were used in this study. Ischemia was produced by the ligation of the left coronary artery for 30 min. Electrocardiogram (ECG), blood pressure, infarct area and blood glucose were determined during the 30 min of ischemia. An arrhythmia score from an ECG recorded during 30 min of ischemia was determined by examining the duration and type of arrhythmia. Different effects of glybenclamide and pinacidil on the arrhythmias were observed in male and female young and middle-age rats. Pinacidil decreased the infarct zone in younger female rats, but differences in the type and length of ischemia-induced arrhythmias between females and males disappeared in older age. The results of this study showed that the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia changed due to the age and gender of rats.