Effect of antioxidants on hypoxia/reoxygenation-induced injury in isolated perfused rat liver.

Isolated perfused livers from rats fasted overnight were subjected to 30 min. of hypoxia followed by reoxygenation for 60 min., resulting in marked cytotoxicity as evidenced by an enhanced release of cytosolic enzymes (lactate dehydrogenase: 14-fold over controls, glutamate-pyruvate-transaminase: 12-fold over controls) and glutathione (twofold over controls) into the perfusate, by calcium accumulation (by a factor of 1.4) in the tissue and by an 80% inhibition of bile secretion. Virtually no mitochondrial injury became apparent and no evidence for lipid peroxidation could be found. In the presence of ascorbate, an augmentation of hepatic injury was observed. This might be due to the pro-oxidant activity of ascorbate in the presence of ionized iron, which is easily released from high molecular weight stores under reductive (e.g. hypoxic) conditions. The water soluble vitamin E analogue trolox C as well as propyl gallate clearly protected the liver against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model. Due to their water solubility and their high efficacy as free radical scavengers, these antioxidants might be of therapeutic value.     

Effects of hypoxia/reperfusion injury on drug disposition in the rat isolated perfused liver

1. Ischaemia-reperfusion injury is known to be associated with a range of functional and structural alterations in the liver. However, the effect of this injury on drug disposition is not well understood. The present study was designed to examine the effects of hypoxia/reperfusion on the disposition of glutamate and propranolol in the rat isolated perfused liver. Both glutamate and propranolol are mainly metabolised in the pericentral region of the liver. 2. Hypoxia/reperfusion was established using the slow flow-reflow method of perfusion in both anterograde and retrograde perfusion. Glutamate metabolism was measured by the recovery of [(14)C]-glutamic acid and [(14)C]-labelled metabolites in a single pass in both anterograde and retrograde perfusion in the presence of a steady state concentration of unlabelled glutamic acid. Propranolol disposition, mean transit time and normalized variance were assessed from the outflow concentration-time profile of unchanged [(3)H]-propranolol determined after a bolus injection of [(3)H]-propranolol using HPLC and liquid scintillation counting. 3. Hypoxia/reperfusion of livers did not affect oxygen consumption, but caused significant changes in enzyme release, lignocaine hepatic availability and bile flow. 4. Hypoxia/reperfusion did not affect the hepatic metabolism of glutamate to carbon dioxide or the hepatic extraction of propranolol. Small but significant changes were evident in the distribution parameters of mean transit time and vascular disposition for the hypoxic-ischaemic liver. 5. It is concluded that reperfusion injury induced by slow flow-reflow perfusion did not influence the extraction of glutamate or propranolol, but may have affected pericentral morphology and solute distribution.     

Cyanide-induced injury to the isolated perfused rat liver

In order to study the events that follow cyanide-induced inhibition of oxidative metabolism and produce cellular injury, isolated, haemoglobin-free perfused rat livers from fasted rats were exposed to KCN (100 mg/l). KCN reduced the oxygen consumption of the livers by about 80%. Hepatotoxicity was evident by a marked release of enzymes (LDH, SDH) and of glutathione (mainly GSSG) into the perfusate, by a depletion of hepatic glutathione and by an accumulation of calcium in the liver. Cyanide-induced hepatotoxicity could be prevented completely by feeding the rats before preparing the liver as well as by addition of fructose to the perfusate of fasted livers. Both treatments resulted in an increased energy supply from anaerobic glycolysis as evidenced by a large release of lactate + pyruvate into the perfusate. The toxic actions of cyanide were markedly attenuated by deferrioxamine as well as by allopurinol. These antitoxic actions occurred without changes in anaerobic glycolysis. Omission of calcium from the perfusate, however, did not influence cyanide toxicity. Thus, energy supply from anaerobic glycolysis seems to be sufficient for the basic functions of the liver to occur, when oxidative metabolism is inhibited by cyanide. The effects of deferrioxamine and allopurinol indicate the involvement of radical intermediates and/or Fe2+ in cyanide-induced cellular toxicity. An influx of calcium from the extracellular to the intracellular space is not involved in cyanide-induced hepatocellular injury.     

Effect of hypoxia on oxidative and reductive pathways of omeprazole metabolism by the isolated perfused rat liver

The effect of hypoxia on the elimination of omeprazole, a potent inhibitor of gastric acid secretion, was studied in the isolated perfused rat liver. During normal oxygenation, a 10 mg bolus dose was eliminated rapidly (T 1/2 beta = 8.0 +/- 1.1 min; mean +/- S.E.M., N = 4), while under hypoxic conditions T 1/2 beta was increased to 81.6 +/- 5.4 min (P less than 0.01). Upon reoxygenation, T 1/2 beta returned to 9.6 +/- 1.3 min. During hypoxia, perfusate concentrations of an oxidative metabolite (the sulphone) were reduced by 68%, while those of the reductively-generated sulphide increased 4-fold. With reoxygenation, both formation and elimination of the sulphone were increased, while the sulphide, which had accumulated during the hypoxic period, was eliminated rapidly. These findings were duplicated in steady-state experiments, in which omeprazole clearance during hypoxia fell by at least 70%, and sulphide concentrations in perfusate rose from undetectable levels to 200 ng/ml (at least a 10-fold increase). Sulphone concentrations did not change with hypoxia, consistent with a reduction in both its formation and elimination rates. We conclude that the hepatic elimination of omeprazole is severely retarded by hypoxia, but that this effect is promptly reversed by reoxygenation. The increased formation of reductive metabolite during hypoxia is not of sufficient magnitude to sustain the normal hepatic elimination of omeprazole.     

Effect of ethanol on uptake of trichloroethylene in isolated perfused rat liver

The effect of ethanol on uptake of trichloroethylene in isolated perfused rat liver was investigated. The uptake of trichloroethylene was measured in both phenobarbital (PB) non-treated and PB treated rat livers. Furthermore, for PB treated rat livers, the fluorescence of reduced pyridine nucleotides, oxygen consumption, and scanning reflectance spectrum were measured in the liver, perfused with Krebs-Henseleit buffer saturated with a 92%O2-5%CO2-3%CO gas mixture. The uptake of trichloroethylene was decreased by 6.0% in the PB non-treated rat liver and 10.6% in the PB treated rat liver following the addition of ethanol. This uptake decrease was thought to arise mainly from the inhibitory effect of ethanol on mixed-function oxidation in the liver because of the corresponding decrease in oxygen consumption and absorbance difference delta A450-490nm. The inhibition was considered to be due to interference with electron transfer to the complex of substrate and cytochrome P-450. Increase in intracellular NADH might also affect the formation of trichloroacetic acid since the reduction of NADH in the cytosol attained a maximum with 20 mM ethanol.     

Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons

Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion (I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1 (TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive cell number and caspase-3 activity. However, pretreatment with Rut (1-10microM) or caspase-3 specific inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons. Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2+ chelator BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2+]i-dependent and PI3K/Akt signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.     

Acute troglitazone action in isolated perfused rat liver

1. The thiazolidinedione compound, troglitazone, enhances insulin action and reduces plasma glucose concentrations when administered chronically to type 2 diabetic patients. 2. To analyse to what extent thiazolidinediones interfere with liver function, we examined the acute actions of troglitazone (0.61 and 3.15 microM) on hepatic glucose and lactate fluxes, bile secretion, and portal pressure under basal, insulin- and/or glucagon-stimulated conditions in isolated perfused rat livers. 3. During BSA-free perfusion, high dose troglitazone increased basal (P < 0.01), but inhibited glucagon-stimulated incremental glucose production by approximately 75% (10.0 +/- 2.5 vs control: 40.0 +/- 7.2 micromol g liver(-1), P < 0.01). In parallel, incremental lactate release rose approximately 6 fold (13.1 +/- 5.9 vs control: 2.2 +/- 0.8 mmol g liver(-1), P < 0.05), while bile secretion declined by approximately 67% [0.23 +/- 0.02 vs control: 0.70 +/- 0.05 mg g liver(-1) min(-1)), P < 0.001]. Low dose troglitazone infusion did not enhance the inhibitory effect of insulin on glucagon-stimulated glucose production, but rapidly increased lactate release (P < 0.0005) and portal venous pressure (+0.17 +/- 0.07 vs +0.54 +/- 0.07 cm buffer height, P < 0.0001). 4. These results indicate that troglitazone exerts both insulin-like and non-insulin-like hepatic effects, which are blunted by addition of albumin, possibly due to troglitazone binding.     

Disopyramide elimination in the isolated perfused rat liver

The elimination kinetics of disopyramide, [14C]disopyramide and [2H]disopyramide have been studied in the isolated perfused rat liver. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0.3-7.5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity. At doses of 0.3 and 7.5 mg the kinetics of [2H]disopyramide showed a small isotope effect probably of negligible importance.     

Disopyramide elimination in the isolated perfused rat liver

1. The elimination kinetics of disopyramide, [¹⁴C]disopyramideand [²H]disopyramide have been studied in the isolated perfused rat liver.

2. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0-3-7-5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition.

3. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity.

4. At doses of 0-3 and 7-5 mg the kinetics of [²H]disopyramide showed a small isotope effect probably of negligible importance.     

Cadmium toxicity in the isolated perfused rat liver

Isolated perfused livers from male and female Sprague-Dawley rats were exposed to cadmium chloride (50 and 200 microM). Acute hepatotoxicity was investigated by measuring cadmium-induced changes in bile flow, urea synthesis and alanine aminotransferase (ALT) leakage. Cadmium-induced lipid peroxidation was estimated by formation of conjugated dieners and thiobarbituric acid (TBA) reactants. Cadmium, at both concentrations, caused a rapid decrease in bile flow (within 40 min) and complete cholestasis within 70 min exposure in livers perfused from both male and female rats. Cadmium exposure (50 and 200 microM) also resulted in the leakage of ALT into the perfusate within 60 min. In contrast, exposure of isolated rat hepatocytes to as high as 500 microM cadmium did not result in enzyme leakage until 180 min exposure. Sex differences in cadmium-induced cholestasis and ALT leakage were not observed at these concentrations. Malondialdehyde was not detected in the perfusate nor were conjugated dienes detected in liver tissue following 90 min cadmium exposure. These data demonstrate that the isolated perfused rat liver (IPRL) is a sensitive system in which to study chemically induced hepatotoxicity. Cadmium rapidly causes functional alterations and cellular damage in perfused livers from both male and female rats. Cadmium-induced liver injury was apparently not related to lipid peroxidation.     

Effect of cadmium on bromosulfophthalein kinetics in the isolated perfused rat liver system.

Bromosulfophthalein (BSP) is a relatively nontoxic organic anion used as an in vivo indicator of liver performance. Elimination of BSP via the biliary system following iv injection requires dissociation from albumin in plasma, translocation across the sinusoidal membrane, conjugation with glutathione within the hepatocyte, translocation across the bile canalicular membrane, and excretion in bile. The effects of cadmium (Cd), anin vivo hepatotoxicant in rats, on BSP kinetics in the isolated perfused rat liver (IPRL) were studied to investigate the interaction between liver toxicity and BSP kinetics. Livers were isolated from male Fisher 344 rats. After a 30-min period for acclimation to the IPRL system, livers were dosed with Cd (as cadmium acetate), in the presence of 0.25% bovine serum albumin, to give initial concentrations of 10 and 100 microM. Sixty min after Cd dosing, the IPRL system was dosed with BSP to give an initial concentration of 150 microM and the elimination kinetics of BSP from the perfusion medium were monitored. Cadmium concentrations in livers at the end of the experiments were 60 +/- 4 and 680 +/- 210 micro mol/kg for the 10 and 100 microM doses, respectively. Exposure to 10 microM Cd for 60 min resulted in a reduction in bile flow, no significant effect on lactate dehydrogenase (LDH) leakage, and slight effects on BSP clearance. Similar studies following exposure to 100 microM Cd showed a dramatic decrease in bile flow with complete cholestasis 60 min after Cd addition. LDH leakage into perfusion medium at the end of the experiment was less than 10%, indicating that Cd affected bile production well before the liver showed significant signs of necrosis. Clearance of BSP from the perfusion medium was dramatically reduced. Taken together, the data indicate that Cd has a significant effect on the kinetics of BSP in the IPRL and the dominant effects were mediated through the cholestatic effect of Cd.     

精胺预处理对离体灌流大鼠心肌缺血/再灌注损伤及细胞凋亡的影响

目的探讨精胺预处理对离体灌流大鼠心肌缺血/再灌注损伤的影响及其可能的抗凋亡作用。方法应用Langehdorff离体灌流大鼠心脏复制模拟心肌缺血/再灌注损伤模型。24只大鼠随机分为3组,每组8只:对照组(Con-trol)、缺血/再灌注组(IR)、精胺干预组(Spermine)。比色法测定冠脉流出液中乳酸脱氢酶(LDH)活力,心肌组织中超氧化物歧化酶(SOD)活性及心肌丙二醛(MDA)含量;多导生理记录系统记录心脏功能;HE染色光镜下观察心肌形态学变化;三苯基氯化四氮唑(TTC)染色检测心肌梗死面积;透射电镜和TUNEL方法检测细胞凋亡;免疫组化检测心肌Fas与Bcl-2蛋白的表达。结果 (1)与对照组比,IR组冠脉LDH漏出明显增多、心肌组织中MDA水平增加、SOD活性降低(P<0.01);心功能明显下降(LVDP,HR,CF均低于对照组,P<0.05);光镜下可见心肌细胞呈凝固性或带状坏死。与Control组比,IR组心肌梗死面积及心肌细胞凋亡率明显增加(P<0.01);心肌Fas蛋白表达上调,Bcl-2蛋白表达下调。透射电镜下心肌细胞核染色质浓缩、凝聚且边集,染色质在核膜周边聚集形成新月形,线粒体嵴排列紊乱。(2)与IR组比,Spermine组冠脉LDH漏出减少,心肌组织中MDA减少,SOD增加(P<0.01);心功能有明显改善(LVDP,HR,CF均明显高于IR组,P<0.05);光镜下心肌细胞结构清晰。Spermine组与IR组比心肌梗死面积及心肌细胞凋亡率均明显降低(P<0.01);心肌Fas蛋白表达下调,Bcl-2蛋白表达上调;电镜下可见心肌肌节结构清晰,线粒体嵴完整、基质致密,未见核染色质凝聚。结论外源性精胺能明显减轻离体灌流大鼠心肌缺血/再灌注损伤,其机制可能与精胺抑制细胞凋亡有关。     

Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung

Acute hypoxia in the intact animal and in cultured endothelial cells has been shown to be associated with a decrease in conversion of angiotensin I (AI) to angiotensin II (AII). Alterations in capillary surface and in contact time resulting from hemodynamic changes have been shown to influence the rate of pulmonary AI conversion. The dependency of AI conversion on hemodynamics complicates the interpretation of experiments showing changes in AI conversion in intact animals. We studied the effect of acute hypoxia on AI conversion in the isolated rat lung perfused at constant flow without recirculation of perfusate. Three levels of oxygenation were produced by ventilating lungs and equilibrating perfusate with a range of hypoxic gas mixtures. AI (1 μg) was injected into the pulmonary artery, and the effluent was collected for measurement of AI and All. Instead of the expected hypoxic inhibition, percent conversion of AI to All increased slightly but significantly from 69.3 ± 3.1 (mean ± S.E.M.) at normal oxygenation to 74.4 ± 3.0 at moderate hypoxia (P < 0.005, paired t) and to 73.5 ± 3.9 at severe hypoxia (P < 0.01, paired t). Decreasing mean transit time of substrate through the lung (by increasing perfusate flow rate from 5 to 20 ml/min) resulted in a significant decrease in conversion of AI from 88.7 ± 2.9 to 73.4 ± 2.1% (P < 0.001, paired t). These data confirm the effect of contact time on the rate of AI conversion in the lungs. The isolated rat lung preparation does not exhibit the phenomenon of hypoxia-induced inhibition of AI conversion. The authors speculate that hypoxia-induced inhibition of AI conversion in vivo may be secondary to the effects of hypoxia on hemodynamics.     

Metabolism of 14C-busulfan in isolated perfused rat liver

1,4-14C Busulfan gave one main metabolite in the isolated perfused rat liver during 4 hr cyclic perfusion. The cumulative bile excretion contained about 38% of the total radioactivity. About 1% of unchanged 14C-busulfan was excreted in the bile. The metabolite was identified as gamma-glutamyl-beta-(S-tetrahydrothiophenium) alanyl-glycine (sulfonium ion of glutathione) by 252Cf-plasma desorption time-of-flight mass spectrometry. The formation of the metabolite was drastically decreased when the glutathione-S-transferase was inhibited, which indicates that the major reaction of busulfan with glutathione is enzymatic in nature. The sulfonium ion was more stable in the perfusate (t1/2 = 22.4 hr, 37 degrees C) than in the bile (t1/2 = 3.2 hr, 37 degrees C) at pH 7.4.     

Effect of chlordecone on carbon tetrachloride-induced increase in calcium uptake in isolated perfused rat liver

Intracellular accumulation of Ca2+ can occur in the livers of animals poisoned with a toxic dose of CCl4. We have reported even greater accumulation of cytosolic Ca2+ in animals treated with an ordinarily nontoxic dose of CCl4 in combination with prior exposure to chlordecone (CD). Present studies were designed to examine if intact perfused livers obtained from animals receiving either CCl4 (100 microliter/kg, ip) alone or in combination with prior dietary exposure to 10 ppm CD for 15 days accumulated 45Ca from the perfusate. Livers obtained at 0, 1, 4, 6, 12, 24, and 36 hr after a single CCl4 injection were perfused with Krebs-Ringer bicarbonate buffer containing erythrocytes, bovine serum albumin, and dextrose. After a 15-min equilibration, 45Ca was added to the perfusate, and the perfusion was continued for 30 min. Hepatic 45Ca accumulation in CCl4-treated animals in the whole liver or in the subcellular organelles obtained from perfused liver was not significantly different from corn oil controls. In the CD + CCl4 combination treatment, 45Ca accumulation followed a biphasic pattern with the first increase at 1 hr and then a progressive rise starting at 12 hr after CCl4 administration. Mitochondrial, microsomal, and cytosolic fractions from perfused liver showed a progressive rise in 45Ca at late time periods, which is indicative of unregulated and irreversible influx of extracellular Ca2+ into these livers. These studies demonstrate that cytosolic Ca2+ progressively increases as a result of the unregulated entry of extracellular Ca2+.     

Metabolism of [14C]monocrotaline by isolated perfused rat liver.

The metabolism of the pyrrolizidine alkaloid [14C]monocrotaline [( 14C]MCT) was examined using the in situ isolated perfused rat liver. Hepatic tissue was perfused in a recirculatory fashion for 90 min and the distribution of metabolites between the bile and perfusate was analyzed. Monocrotalic acid (MCA) was found to be the major acidic metabolite of [14C]MCT, with trace amounts of 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and 1-hydroxymethyl-7-oxo-6,7-dihydro-5H-pyrrolizine (tentative assignment) being identified in the perfusates using GC/MS. MCT N-oxide was also identified but represented less than 4% of the perfusate 14C. The simple necine base retronecine was not present at detectable levels in the perfusion medium. A large portion of the 14C recovered from both the bile and perfusate was not extractable, under acidic or basic conditions, into organic solvents. Using fast atom bombardment MS/MS, a portion of this material was identified as a glutathione conjugate of DHP. In addition, this nonextractable material retained a portion of the radioactivity that was equivalent to the acidic fraction. Given these findings and the absence of retronecine, the major pathway for the metabolism of MCT could potentially involve the production of MCT pyrrole, which subsequently reacts with cellular nucleophiles producing MCA in addition to highly water-soluble conjugated pyrroles and possibly macromolecular adducts.