| BackgroundThe cardiovascular toxicity of anticancer agents can lead to serious complications in tumor patients and impact their long-term survival quality. Many cancer survivors will actually be at as great a risk from the involved cardiac disease as from the recurrent of cancer. Some necessary therapies for them may be encumbered with their cardiovascular complications. Therefore, the antitumor diorganotin compounds with relative low toxicity have been paid more attentions. Dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) is a new dioganotin (Ⅳ) arylhydroamate complex with 4-chlorobenzohydroxamic acid as a ligand which shows high antitumor activity in vivo and in vitro. However, it still does not get into clinical trail due to the remaining necessary researches and its toxicity on cardiovascular system which have ever not evaluated yet. In this research, the toxic effects of dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) as a organotin compounds on cardiovascular system and the mechanisms underlying them were studied.Various factors were implicated in the cardiovascular toxicity of antitumor agents. In general, free radical induced by oxidative stress, calcium overload and mitochondrial damage were to be the crucial events and they formed a network.with action each other, which aggravated further the damage to heart and vessels. Among them, the intracellular calcium overload was considered as a pivotal and sensitive event to exogenous compounds invasion, being the common pathway of cell damage and death.The changes of cardiac function and vascular tone were related closely with intracellular calcium concentration and could be as the sensitive index for abnormal calcium handling. During our pilot experiment, we found that DBDCT inhibited cardiac function and deteriorated particularly cardiac diastolic function and aggravated continuously after washing that, showing an irreversible change. In the meantime, DBDCT caused evident increase of the coronary flow, suggesting its relaxed action on coronary. The mentioned results mean that DBDCT induced salient changes of calcium handling in myocardium and vascular smooth muscle, which may be the pivotal step in its toxic effects on heart and vessels. In this study, our work would focus in calcium handling and relative events underlying it in order to explore the toxic effects of DBDCT on cardiovascular system and analysis their mechanisms. The cardiac function in vivo and in vitro, biochemical index, tissue morphology, myocardial ionic currents and intracellular calcium concentrations were observsed. We also observsed the changes of the coronary flow and the tone of aortic rings induced by DBDCT, combinating with the ion channel blockers to search for mechanism of them.The study is divided into three parts as below.Objective:In order to confirm the effects of DBDCT at different concentrations on rat heart we observsing the changes of cardiac function, biochemical index and morphology structure.Methods:1. Preparation of acute administrationAfter the adult Sprague-Dawley rats were anesthetized, left ventricular cannulation was performed to detected the cardio funtion indexes such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax, and the standard limbⅡ-leads of the ECG were recorded. All of the above indexes were recorded for 2 h before and after administrated (intraperitoneal injection) DBDCT or vehicle or saline. Serum samples were collected for detection of enzyme targets.The experimental groups were divided as follows:1) The control group:intraperitoneal injection at 5.0 mg/kg dose of the same volume of saline.2) The vehicle group:intraperitoneal injection at 5.0mg/kg dose of the same volume of vehicle.3) DBDCT 2.5 mg/kg group:intraperitoneal injection of 2.5 mg/kg DBDCT.4) DBDCT 5.0 mg/kg group:intraperitoneal injection of 5.0 mg/kg DBDCT.2. Preparation of chronic administrationHealthy adult SD rats were randomly divided into experimental and control group, intraperitoneal injection every other day. Left ventricular systolic pressure (LVSP). left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax. the standard limbⅡ-leads of the ECG. body weight (BW), ventricular weight (VW), and zymologic index of myocardial tissue, histological changes were observed at the first 10 days and the first 20 days after administration of drugs.The rats were randomly divided into 6 groups as follows:1) The control group:intraperitoneal injection the same volume saline of 5.0 mg/kg 5 times and 10 times every other day.2) The vehicle group:intraperitoneal injection the same volume vehicle of 5.0 mg/kg 5 times and 10 times every other day.3) DBDCT 2.5 mg/kg 10 days group:intraperitoneal injection 2.5 mg/kg DBDCT 5 times every other day.4) DBDCT 5.0 mg/kg 10 days group:intraperitoneal injection 5.0 mg/kg DBDCT 5 times every other day.5) DBDCT 2.5 mg/kg 20 days group:intraperitoneal injection 2.5 mg/kg DBDCT 10 times every other day.6) DBDCT 5.0 mg/kg 20 days group:intraperitoneal injection 5.0 mg/kg DBDCT 10 times every other day.Results:1. The acute effect of DBDCT on heart in vivo1) A slightly enhanced cardiac function was observed during administrating DBDCT at the low dose (2.5mg/kg), showing a slight increase in left ventricular systolic pressure (LVSP), left ventricular developed pressure (LVSP-LVEDP) and+dp/dtmax. In contrast, above parameters were lower significantly than vehicle group in high dose (5.0mg/kg) group, showing obvious decrease of cardiac function. Noteworthy is the left ventricular end diastolic pressure (LVEDP) in both two groups were increased though have no statistics significant vs vehicle group.2) After administrating 2.5 mg/kg and 5.0 mg/kg DBDCT intraperitoneally, the changes of zymologic index are following:lactate dehydrogenase (LDH) content in serum were increased to (722±86) U/L and (872±106) U/L(P<0.01), respectively; creatine kinase (CK) and malondial-dehyde (MDA) increased slightly; superoxide dismutase (SOD) decreased slightly (P>0.05).3) Change of ECG showed occasionally the premature ventricular contractions after administrating 5.0 mg/kg DBDCT trapentoneally.2. Chronic effect of DBDCT on heart in vivo1) The index of cardiac function generally increased by low dose (2.5 mg/kg) DBDCT, but the LVEDP in 10 days and 20 days have no significant change. In contrast, the cardiac function is significantly inhibited and is getting deterioration with time by high dose (5.0 mg/kg) DBDCT. All index have significant differences comparing with vehicle group besides the LVSP at the first 10 days, indicating the decrease of left ventricular developed pressure (LVSP-LVEDP) mainly related to the increasing LVEDP.2) Serum lactate dehydrogenase (LDH) caused by DBDCT 2.5 mg/kg 10 days and 5.0 mg/kg 10 days were significantly rised from (564±153) U/L to (900±103) U/L(P<0.01) and (912±88) U/L (P<0.01), respectively, and keeped at this level in the twentieth day. DBDCT caused increase of tissue malondialdehyde (MDA) mainly in the early stages after administrating DBDCT from (59±13) nmol/mgprot to (110±37) nmol/mgprot (2.5 mg/kg 10 days) (P<0.05) and (112±33) nmol/mgprot (5.0 mg/kg 10 days)(P<0.05). But it could recovery to the level before administration at the twentieth days.3) Increase of the voltage and the heart rate inⅡlead ECG can be observed at 2.5 mg/kg 20 day group, and the remaining groups had no significant change in ECG.4) There were no significant changes of VW/BW in all groups of animal. Pathological examination demonstrated that the myocardial tissue have also no obvious abnormal. Occasionally we can see infiltration of inflammatory cells at 5.0 mg/kg 20 days group.Objective:To explore the mechanisms of myocardial toxicity by DBDCT, we observed the changes of cardiac function in vitro, myocardial ion currents and intracellular calcium concentration after administration of DBDCT.Methods:1. Preparation of perfused heart of rat in vitroAfter heparinization and anesthetization, the rat hearts were quickly removed and mounted on Langendorff aortic retrograde perfusion system. Water balloon that connectioned with the biological signaling recording and analysis system of Powerlab and Chart 6.0 Data was put into the left ventricular to detect the cardio funtion indexes such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP). left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax and coronary flow was recorded. Water balloon maintained left ventricular end diastolic pressure (LVEDP) at 2-10mmHg. Left ventricular developed pressure (LVSP-LVEDP) greater than 60mmHg and HR>200 times/min were considered the steady condition of the heart. After 30 min equilibrium, administrated drugs for 30 min, and then perfusion Tyrode's solution 30 min. Perfusate and heart tissue were collected to observe zymologic index and histological changes.The rats were randomly divided into 7 groups as follows:1) The control group:perfusion with Tyrode's solution for 1.5 hours.2) The vehicle group:administerate the same volume of vehicle as 1×10-5M in the Tyrode's solution.3) DBDCT 5×10-6M group:administerate 5×10-6M DBDCT in the Tyrode's solution.4) DBDCT 1 x 10-5M group:administerate 1×10-5M DBDCT in the Tyrode's solution.5) L-NAME group:administerate L-NAME (100μmol/L) in the Tyrode's solution.6) L-NAME group+DBDCT 5×10-6M group:administerate L-NAME (100μmol/L) and 5×10-6M DBDCT in the Tyrode's solution.7) L-NAME group+DBDCT 1×10-5M group:administerate L-NAME (100μmol/L) and 1×10-5M DBDCT in the Tyrode's solution.2. Record the effects of ion channel and transporter.Single rat ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase. Using whole cell recording and voltage-clamp mode, the effects of different dose DBDCT (10-7M,1×10-6M,3×10-6M,5×10-6M,8×10-6M,10-5M)on ICa,Ito,INa and INa/Ca, were recorded.3. Calcium transient and resting calcium concentration determination in rat ventricular myocytesSingle rat ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase, then staining with Fluo-3 AM or Fluo-4 AM. Using IonOptix single cell synchronous motion edge detection system to monitor cell synchronization systolic and diastolic function and calcium transient; At a condition of calcium Tyrode's solution and calcium-free Tyrode's solution, we observed the changes in fluorescence intensity caused by DBDCT 1×10-5 and with the intervention of sarcoplasmic reticulum RyR blocker ryanodine 100μmol/L and sarcoplasmic reticulum calcium pump inhibitor thapsigargin 5μmol/L in the laser confocal microscope view.Results:1. In the isoladed and perfused heart, DBDCT inhibited cardiac function significantly with a concentration dependence, especially on diastolic function. The rise of LVEDP is the most salient and sensitive, so that the arrest in contracture could be observed in high concentration groups. In addition, during the period of washout of drug, the cardiac function was deteriorated continuously rather than recovered.2. LDH in perfusate of low concentration (5×10-6M) and high concentration (1×10-5M) DBDCT were significantly increased from (43±5) U/L to (127±20) U/L and (111±11) U/L, respectively. After washout of drug, LDH was reduced, but still significantly higher than that before administering (P<0.01).3. Histopathological examination revealed that DBDCT caused obvious pathological changes of myocardium, including nuclear loss and color inequality.4. DBDCT could increase coronary blood flow in vitro. Under constant pressure perfusion, DBDCT 5×10-6M and 1×10-5M increased coronary flow before the administration from (12.2±0.7) ml/min to (22.9±1.5) ml/min and (24.0±1.5) ml/min, respectively. It continues to increase coronary flow to (24.8±1.1) ml/min and (24.6±1.1) ml/min after washing drug with Tyrode's solution.Combining application of nitric oxide synthase inhibitor L-NAME and DBDCT 1×10-5M caused the coronary blood flow increased to (25.4±1.1) ml/min that is the same level as the DBDCT did alone. It suggested that the increase in coronary flow induced by DBDCT was mainly result from the directly relaxing effect of drug on vascular smooth muscle.5. DBDCT at 5×10-6M,8×10-6M and 10-5M could concentration-dependently decreased ICa-L and Ito. DBDCT at 5×10-6M,8×10-6M and 10-5M decreased the current density of ICa-L before the administration from 41.75±4.60 (pA/pF) to 27.57±4.83 (pA/pF) (33.75%),21.82±4.88 (48.54%) (pA/pF) and 17.56±4.34 (58.71%) (pA/pF) (P<0.05), and made the based current density of Ito decrease of 49.56%,72.42%和73.15%(P<0.05). Both of ICa-L and Ito were not recovered after washing 3 min.Meanwhile, DBDCT at different concentration have no effect on INa and INa/Ca.6. DBDCT can significantly increase the resting intracellular calcium concentration of cardiac myocytes in both calcium-containing and calcium-free perfused solution. DBDCT 10-5M in the calcium-containing solution 5 min and 15 min could increase calcium concentration of (16.39±2.71)% and (23.55±1.29)%(P<0.05), respectively; DBDCT 10-5 M in the calcium-free conditions 5 min and 15 min could increased intracellular calcium concentration of (4.83±2.02)% and (23.55±2.20)%(P<0.05). This results suggesting that the increase intracellular resting calcium concentration by DBDCT was independent of extracellular Ca2+ Meanwhile, the increase of intracellular calcium concentration induced by DBDCT could be preventing completely by RyR blocker ryanodine 100μmol/L, which suggesting the increase of intracellular calcium concentration by DBDCT depends on the role of ryanodine receptors. Further investigation showed that in calcium-free extracellular solution, adding thapsigargin, a sarcoplasmic reticulum (SR) calcium pump inhibitor, and 15 min could enhance the increased effects of DBDCT on intracellular calcium concentration. Combining applycation of 10-5M DBDCT and 5μM thapsigargin at 5 min after administering could double the effects of DBDCT alone (9.52% vs.4.83%). This result suggests that SR calcium pump might be excited by DBDCT as well. Furthermore, the exciting effect of DBDCT on SR calcium pump showed a time phase that is stronger in the early time (5 min) but weaker in the late time (15 min). This conclusion was supported by the fact that DBDCT in the calcium-free solution containing both ryanodine and thapsigargin could decline the intracellular calcium concentration (-14.38±5.34)% at 5 min after administering while elevate it (15.71±4.81)% at 15min after administering.7. DBDCT significantly inhibited the calcium transient and contraction of myocardial cells. The peak of calcium transient in myocardial cells is reduced to (90.64±2.97)% of baseline and the amplitude of cell contraction was decreased to (80.42±2.52)% of baseline after administering DBDCT 10-5M.Objective:The study was designed to observe the impact of dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) to aortic rings to investigate the effects and the possible mechanism(s) of DBDCT on isolated thoracic aorta rings.Methods:The rat was sacrificed by cervical vertebra luxation and the thoracic aorta was quickly isolated from each animal and placed in cold physiological salt solution (PSS) to be used in vascular tone of isolated thoracic aortic experiments. Isotonic tension of thoracic aortic rings pre-contracted by KCl (30 mmol/L) or NE (10-6 mol/L) was recorded. The relaxant effects of DBDCT in 5×10-6 mol/L,1×10-5 mol/L and effects of various drugs include endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME.10-4 mol/L), cyclooxygenase inhibitor indomethacin (Indo, 10-5mol/L), KCa channel blocker tetraethylammonium (TEA, 1×10-2 mol/L). KA channel blockers 4-aminopyridine (4-AP, 10-5mol L), were observed in the rings. The influence of KATP channel blocker glibenclamide (Gli,1×10-5 mol L), barium chloride (BaCl2,10-5 mol/L) on the effects of DBDCT were also studied. Results:1. DBDCT (5×10-6 M,1×10-5 M) concentration-dependently attenuated the contraction induced by KC1 (60 mmol/L) and NE (1×10-6 mol/L) in intact endothelium and denuded endothelium of isolated thoracic aorta. There was significant difference in the results campared with vehicle group (P<0.01), but in that between the rings with intact and denuded endothelium at the concentrations of DBDCT (P>0.05).2. The relaxant effects of DBDCT have no significant difference between pretreated and untreated with L-NAME, Indo, Gli, and 4-AP (P>0.05). The relaxant effects of DBDCT on vascular rings were attenuated by pretreated with TEA, BaCl2(P<0.05).3. DBDCT 5X 10-6mol/L and the vehicle were administrate in the calcium-free PSS solution, after 20min incubation bath, a rapid and short-term contraction of vascular ring were produced by NE 10-6 mol/L, the percentage of NE contraction in vehicle group and DBDCT 5×10-6M group was (20.44±1.60)% and (1.89±0.32)%, respectively. The amplitude inhibited by DBDCT 5×10-6mol/L group was significantly larger comparing with that by vehicle group (P<0.01). The contraction of vascular ring was produced again by adding CaCl2 2.5 mmol/L in the same condition, the percentage of contraction produced by CaCl2 in vehicle group and DBDCT 5×10-6 mol/L group were (91.87±2.16)% and (1.60±0.48)%, respectively. The amplitude inhibited by DBDCT 5×10-6 mol/L group was significantly larger comparing with that by vehicle group (P< 0.01). The results showed that DBDCT (5×10-6 mol/L) inhibited the contractions induced both by NE-induced calcium release and calcium influx.Conclusion:1. dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) displays a dose and time-dependent toxicity on the rat heart, including gradually aggravating deterioration of the cardiac function, especially in diastolic function, the increase of LDH and MDA, and inflammatory infiltration of myocardial tissue. However, its toxicity is still lighter comparing with that of doxorubicin in the existing document.2. ICa-L was inhibited by DBDCT dose-dependently, while the sarcoplasmic reticulum ryanodine receptor can be intensely excited, which may be responsible for the persistent elevation of intracellular calcium and geting deterioration of diastole.3. DBDCT can significantly increase the coronary flow. This effect was not inhibited by L-NAME. indicating that the drugs may have a direct relaxation on coronary artery smooth muscle.4. DBDCT attenuated the contraction induced by KCl and NE in intact endothelium and denuded endothelium of isolated thoracic aorta. The relaxtion of thoracic aortic rings by DBDCT were independent of endothelium as well as the NO and PGI2 released by it. This effect may be a result from a direct action on vascular smooth muscle and implicate the activation of KiR and KCa currents and the inhibition of calcium influx and sarcoplasmic reticulum calcium release.
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