The influence of oxygen tension on theophylline clearance in the rat isolated perfused liver

The effect of changes in the rate of perfusion and oxygen tension (PO2) on theophylline clearance was determined in the rat isolated perfused liver. Changes in rate of perfusion had no effect on the extraction ratio of theophylline while a decrease in the PO2 significantly increased the half life of theophylline. These results suggest that changes in PO2 whether due to disease states or oxygen administration may necessitate theophylline dosage adjustment.     

氯丁二烯吸入染毒致大鼠急性肝损害的氧化应激机理

大鼠吸入浓度为6.08 mg·L~(-1)氯丁二烯4 h后。2,12和24 h处死.染毒后2 h时血清SDH即明显升高,继而ALT升高,前者增高的幅度大于后者,肝匀浆GSH明显下降,然后升高,脂质过氧化产物MDA形成增多,肝线粒体膜脂流动性下降,与线粒体膜结合的ANS荧光增强,表明氯丁二烯所致急性肝损害可能与氧化应激机理有关。     

Bradykinin inactivation by perfused rat liver

Summary Previous data had suggested the presence of at least two enzymes in the perfused rat liver — a peptidyldipeptide hydrolase (EC 3.4.15.1) which inactivates bradykinin (BK) and converts angiotensin I (AI) to angiotensin II (AII), and an enzyme which inactivates AII. However, in the present studies the amides of BK and bradykinylglycine were inactivated by the perfused liver at the same rate as BK suggesting that the inactivation of BK was due not only to the presence of the peptidyldipeptide hydrolase which requires a free carboxyl group but to an additional, as yet unidentified kininase. A 10-min perfusion of rat liver with oxygenated Tyrode solution containing 0.05% (v/v) Triton X-100 liberated significant amounts of kininase, potassium, lactic dehydrogenase and acid phosphatase into the perfusion medium.DEAE-cellulose chromatography of this perfusate purified the kininase activity, which does not hydrolyze either AI or AII. BK hydrolysis at pH 7.0 by this enzyme was inhibited by 1×10^–3 M sodium tetrathionate but not by 3×10^–3 M EDTA or BPP_9a (10 g/ml), a BK-potentiating nonapeptide which inhibits the peptidyldipeptide hydrolase. The endopeptidase splits BK between the Phe⁵-Ser⁶ bond forming the peptides Arg¹-Phe⁵ and Ser⁶-Arg⁹ in stoichiometric amounts. It may be crucial for the BK inactivation by the perfused rat liver.Work supported by grants from FINEP, Rio de Janeiro and FAPESP, São PauloFrom Department of Medicine, EPMResearchers from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Rio de Janeiro.     

Reductive drug metabolism in isolated perfused rat liver under restricted oxygen supply

1. Hepatic azo and nitro reductase activities were studied in the perfused rat liver under normal and restricted oxygen supply. 2. Formation of sulphanilamide or p-aminobenzoic acid from neoprontosil or p-nitrobenzoic acid under aerobic conditions of liver perfusion was negligible, even at a reduced oxygen saturation of a pO2 of 300 mm Hg in the haemoglobinfree perfusion system. At a pO2 of 200 mm Hg reductase activities were almost maximal. 3. Conjugation of sulphanilamide (0-08 mM) was similar under aerobic and anaerobic conditions. Hepatic elimination of p-aminobenzoic acid (0-08 mM) showed an oxygen-dependent increase for 15 min after addition of substrate. 4. p-Nitroanisole demethylation was inhibited 80% under hypoxic perfusion at 200 mm Hg pO2 and was completely inhibited after gassing with anoxic mixtures. 5. Restitution of aerobic conditions after 30 min anaerobic perfusion restored hepatic respiration, lactate pyruvate ratio, and pH value to levels found under aerobic conditions, but bile flow remained 50% reduced.     

Sex differences in sulfobromophthalein-glutathione transport by perfused rat liver

Sex differences have been described in the hepatic transport of many organic anions. Proposed mechanisms include differences in the rate of metabolism, in the degree of binding to cytoplasmic proteins, and in the rate of membrane transport. To better define these factors, we used the perfused rat liver to study the hepatic transport of the glutathione conjugate of sulfobromophthalein (BSP-GSP), a model compound that does not require metabolism for excretion. Hepatic transport of BSP-GSH was saturable for both sexes. Clearance of BSP-GSH from 1% albumin solutions at steady-state was 35-52% greater in female livers than in male livers, and reflected a 47% larger apparent Vmax with no change in the apparent Km. Analysis of the rate of disappearance of BSP-GSH from recirculating perfusate and its appearance in bile using a simple two-compartment model indicated that the ratio of influx to efflux was greater in female livers. These findings are compatible with sex-related differences in the electrochemical driving forces for BSP-GSH uptake.     

Oxidative stress in rat brain but not in liver following oral administration of a low dose of nanoparticulate silver

While it is known that silver nanoparticles (AgNPs) can enter the brain, our knowledge of AgNP-induced neurotoxicity remains incomplete. We investigated the ability of 10 nm citrate-stabilized AgNPs to generate oxidative stress in brain and liver of adult male Wistar rats after repeated oral exposure for 14 days, using a low dose of 0.2 mg/kg b.w. as compared with the same dose of ionic silver (silver citrate). In AgNP-exposed animals, the level of reactive oxygen species (ROS), lipid peroxidation (MDA) and glutathione peroxidase (GPx) activity were found to be significantly higher in brain relative to the control group receiving saline. Administration of ionic silver (silver citrate) increased ROS and MDA levels in both tissues. Activities of GPx in brain so as superoxide dismutase (SOD) and catalase (CAT) in liver of exposed animals were also elevated. Besides, AgNPs and silver ions were both found to cause statistically significant decrease in the reduced-to-oxidized glutathione ratio (GSH/GSSG) in brain. The results show that exposure to a very low dose of particulate silver generates mild oxidative stress in the brain but not in the liver of rats, indicating a role of oxidative stress in AgNP-induced neurotoxicity.     

Catabolism of vasoactive polypeptides by perfused rat liver

Summary Exsanguinated rat liver preparations perfused in situ with oxygenated saline solutions inactivated recirculating bradykinin (BK) at rates of 2.3 to 9.1 and isoleucyl⁵ angiotensin II (AII) at rates of 2.8 to 15.0 nmolex x min^–1 x g^–1 of liver, depending on the initial concentration of the peptides in the perfusion fluid (3.1 to 18.9×10^–6 M for BK and 8.5 to 17.0×10^–6M for AII). On the other hand, at similar concentrations, recirculation of isoleucyl⁵ Angiotensin I (AI) for 8 min did not lead to decrease of its biological activity when assayed on the isolated rat uterus. Following a single passage through liver, picomole amounts of both BK and AII were inactivated by about 90% as revealed by assays on a superfused rat uterus.The potency ratio AI:AII, assayed on a superfused rat uterus was 1:22 and changed to 1:5 following a single passage of both peptides through liver. This finding and the separation of 4.9% of AII on carboxymethylcellulose columns following recirculation of AI through rat liver indicate a conversion of AI into AII. The dipeptides Phe-Arg, Ser-Pro and Gly-Phe were identified among the hydrolysis products of perfused BK. A peptidyldipeptide hydrolase (EC 3.4.15) may be responsible for both the BK inactivation and AI conversion. The inactivation of AII cannot be attributed to the same enzyme.Abbreviations BK bradykinin - AII isoleucyl⁵ angiotensin II - AI the corresponding angiotensin I - BPP 5a bradykinin-potentiating peptide PCA-Lys-Trp-Ala-Pro Work supported by grants from Project BIOQ/FAPESP (São Paulo) and FINEP (Rio de Janeiro)From Department of Medicine, EPMFrom Department of Physiology and Biophysics, EPM     

Nitrofurazone: kinetics and oxidative stress in the singlepass isolated perfused rat liver

The disposition of the antibiotic nitrofurazone was studied in the singlepass isolated perfused rat liver. Both the effects of the steady-state level of drug and the composition of the perfusate were evaluated. The higher level (120 micrograms/ml) of nitrofurazone in a perfusion medium lacking the glutathione (GSH) precursors, glycine, glutamic acid and cysteine, caused a marked increase in bile flow (from 1.01 +/- 0.07 to 2.33 +/- 1.07 microliters/min/g), massive biliary efflux of glutathione disulfide (GSSG) (from 0.55 +/- 0.07 to 60.6 +/- 25.4 nmol/min/g) and a sharp decline in the caval efflux of GSH (to undetectable levels) and the tissue level of GSH (from 5.74 +/- 0.20 to 2.68 +/- 0.13 mumol/g). Even after the drug was discontinued, these parameters were not restored to control levels. The lower level (30 micrograms/ml) of nitrofurazone with or without amino acid supplementation and the higher level with supplementation induced less dramatic effects. Using [35S]methionine, a new conjugated metabolite of nitrofurazone and glutathione was detected. The data suggest that the toxicity of the reactive oxygen species generated by the redox cycling of the nitro group and the reactive metabolites generated by further reduction of nitrofurazone can be mitigated by adequate glutathione levels, but that livers lacking sufficient glutathione to scavenge these reactive species may be damaged.     

Ethanol potentiates oxygen uptake and toxicity due to menadione bisulfite in perfused rat liver

Menadione bisulfite is a hepatotoxicant that damages periportal regions of the lobule in perfused liver in an oxygen-dependent manner. The effect of ethanol on menadione bisulfite toxicity was examined in perfused rat liver. Addition of menadione bisulfite (3 mM) alone to the perfusate increased oxygen uptake by 20-30 mumols/g/hr. Lactate dehydrogenase was released into the effluent after 60 min of perfusion and reached values around 100 units/g/hr. Under these conditions, trypan blue was taken up exclusively in periportal regions of the liver lobule; 44% of periportal cells were stained. In the presence of ethanol, maximal increases in oxygen uptake due to menadione bisulfite were much larger (about 90 mumols/g/hr), and lactate dehydrogenase release occurred earlier and reached higher maximal values (330 units/g/hr). Trypan blue staining was also more extensive; 90% of periportal cells were stained. The effect of ethanol on menadione bisulfite-induced oxygen uptake required metabolism via alcohol dehydrogenase (ADH), because ethanol increased oxygen uptake due to menadione bisulfite from 44 to 81 mumols/g/hr in deermice with ADH but had no effect in deermice lacking ADH. Other agents that increase NADH (xylitol and 2-ethyl-1-hexanol) also potentiated the stimulation of oxygen uptake due to menadione bisulfite, suggesting that ethanol was working by increasing the NADH redox state. Cyanide abolished the increase in oxygen uptake due to menadione bisulfite, both in the absence and in the presence of ethanol, supporting the hypothesis that the effect of ethanol on menadione bisulfite-mediated oxygen uptake involves the mitochondrial respiratory chain. Further, the stimulation of oxygen uptake by menadione bisulfite in isolated mitochondria was enhanced when matrix NADH was increased by addition of beta-hydroxybutyrate. These data indicate that ethanol potentiates oxygen uptake and toxicity due to menadione bisulfite most likely by generation of NADH for redox cycling of this model quinone.     

丙烯腈对大鼠脑皮层氧化应激效应的研究

目的　评价长期低浓度接触AN对大鼠脑组织的氧化性应激效应 ,探讨AN神经毒性机制。方法　SD雄性大鼠 3 0只 ,随机分成 3组 :对照组、低剂量组和高剂量组 ,通过饮水分别给予 0、5 0和 2 0 0mg L的AN溶液。染毒时间为 12周。染毒结束后从每组随机选取 7只大鼠 ,取双侧大脑皮层测定氧化性应激指标。结果　染毒组的MDA水平(nmol mg蛋白 )明显高与对照组 (16 60± 6 49) ,且高剂量组 (15 7 7± 85 40 )明显高于低剂量组 (98 5 3± 5 0 3 5 ) ,其剂量 效应关系有显著性 ;3组之间的GSH Px活力差异未见显著性 ;虽然低剂量组的SOD活力和对照组的差异未见显著性 ,但高剂量组明显高于对照组和低剂量组。高剂量组GSH明显低于对照组和低剂量组 ,但低剂量组和对照组的差异无显著性。结论　诱导氧化性应激可能是AN神经毒性的重要作用机制。     

Effect of oxygen tension on the generation of alkanes and malondialdehyde by peroxidizing rat liver microsomes

The alkanes, ethane and pentane, are often used as indices of lipid peroxidation. Because it has been indicated that O2 tension can affect the yield of these compounds, a systematic study of this was carried out. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. Ethane and pentane production were measured by gas chromatography, and malondialdehyde was measured by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Polyunsaturated fatty acids were lost during lipid peroxidation. There was no loss of saturated or monounsaturated fatty acids. Loss of polyunsaturated fatty acids correlated with O2 tension in the flask. Half-maximal losses of docosahexaenoic acid, arachidonic acid, and linoleic acid occurred at 3, 5, and 35% O2 respectively. Malondialdehyde formation reflected polyunsaturated fatty acid loss at all O2 concentrations. Alkane formation reflected polyunsaturated fatty acid loss below 5% O2 but not above it. The ratio of alkane formed to precursor polyunsaturated fatty acid lost decreased progressively as O2 concentration was increased above 5%. For example, the molar yield of pentane formed per precursor polyunsaturated fatty acid lost was 0.3% at 5% O2 but only 0.003% at 100% O2. This indicates that quantitation of lipid peroxidation using alkane formation requires consideration of O2 tension at the site of alkane formation.     

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by oleate

p-Nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats was rapidly and reversibly inhibited by sodium oleate (0.3 to 0.6 mM). Xylitol partially reversed this inhibitory effect. The inhibition was not mediated by a direct effect of oleate on microsomal components since concentrations of oleate ranging up to 1.0 mM did not affect p-nitroanisole O-demethylation by isolated microsomes. Infusion of 0.6 mM oleate did not alter the measured intracellular NAD+/NADH ratio but did cause a significant increase in the intracellular NADP+/NADPH ratio. A significant decrease in the ATP/ADP ratio was also observed. Oleoyl CoA inhibited p-nitroanisole O-demethylation in microsomes (Ki about 30 microM), and both oleoyl CoA and palmitoyl CoA inhibited the energy-linked nicotinamide nucleotide transhydrogenase in submitochondrial particles (Ki about 1 microM). Thus, inhibition of mixed-function oxidation in the intact liver by oleate is most likely mediated by oleoyl CoA. Oleoyl CoA inhibits mixed-function oxidation in the intact liver by acting directly on cytochrome P-450 and by decreasing generation of NADPH via inhibition of key enzymes of the citric acid cycle and the energy-linked transhydrogenase.     

Inhibition of gluconeogenesis in isolated perfused rat liver by clanobutin

The effect of clanobutin {4-[p-chloro-N-(p-methoxyphenyl-)benzamido]butyric acid} on gluconeogenesis from lactate + pyruvate (1.6 + 0.2 mmoles/1) as precursors in isolated perfused liver of fasted rats was investigated. Glucose production was dose dependent inhibited up to a maximum of 81 ± 3 per cent; the half-maximum concentration of clanobutin was 0.33 ± 0.04 mmoles/1. Buformin and phenformin, respectively, used as references showed no effect on gluconeogenesis under the given experimental conditions. Oxygen uptake was not inhibited by clanobutin at concentrations up to 0.14 mmoles/1. At higher concentrations, the inhibitory effect was smaller than observed for comparable buformin doses. According to our results, clanobutin appears to be a more potent and probably more specific inhibitor of gluconeogenesis than the therapeutically used biguanides—at least in the isolated perfused rat liver.     

Microsomal enzyme activity in perfused rat liver

The activities of microsomal enzymes were observed during perfusion of livers isolated from both normal and phenobarbital treated rats. The hydroxylation of aniline and the O-demethylation of p-nitroanisol did not decrease substantially (by 10 per cent only) in the 9000 g fraction prepared from livers perfused for 4 hr.N-demethylation of aminopyrine was reduced by 60 per cent in the same experimental conditions. A similar decrease of N-demethylation was also observed when livers were isolated from animals treated with phenobarbital. The content of cytochrome P-450 in the microsomes was stable during a 4-hr perfusion of isolated liver. The decrease of N-demethylation of aminopyrine could be partly restored by the simultaneous infusion of nicotinic acid, which is a precursor of NAD and NADP biosynthesis.     

Prilocaine elimination by isolated perfused rat lung and liver

Prilocaine is assumed to undergo significant elimination by extrahepatic organs and to differ in this respect from other commonly used local anaesthetics. In order to clarify whether the lung may play an important role as a site of elimination of prilocaine, the kinetic parameters were studied in isolated perfused rat lungs and were compared to those of isolated livers. Furthermore, the structurally related compounds bupivacaine and mepivacaine were also investigated in this system. Prilocaine was dispersed into a relatively large apparent distribution volume in perfused rat lung (139 ml versus 97 ml in controls). In single-pass perfused lungs the observed maximum of concentration was decreased by about 60% compared to controls. The mean residence time was prolonged by about 40%. These observations suggest that prilocaine is substantially retained by rat lung and that this effect occurs particularly during first-pass.However, the ability of rat lung to degrade prilocaine was relatively low. The clearance values were about 0.3 ml/min equal to about 20% of the hepatic capacity calculated per g of tissue. Thus it must be assumed that prilocaine is only transiently retained by the lung and will gain systemic availability later on. In rat lungs the kinetics of prilocaine elimination were not substantially different from those of bupivacaine and mepivacaine (16 and 12%). These observations do not support the assumption that especially prilocaine undergoes extrahepatic elimination.For low (2 g/ml) and intermediate (10 g/ml) drug concentrations isolated rat liver exhibited clearance values close to the perfusion flow rate. Accordingly, prilocaine was removed from the perfusion medium of isolated livers already during first-pass. At very high concentrations of 100 g/ml, the clearance dropped to about half of the control values. Thus under these conditions approximately half of the dose escaped first-pass extraction which is probably caused by saturated metabolic clearance. Such an effect was not observed for bupivacaine and mepivacaine.Part of the doctoral thesis of Markus Ebke, Medical Faculty of the University of Göttingen     

Prostaglandin uptake and metabolism by the perfused rat liver

1. The prostaglandins are C20 unsaturated fatty acids which exhibit diverse physiological effects of short duration. We have investigated the speed of removal of PGE₁ and PGF_1α from the circulating blood and their subsequent metabolism by the isolated perfused rat liver.

2. Following either a single injection of radiolabelled PGE₁ or PGF_1α into the hepatic artery or portal vein, or recirculation of prostaglandins through the liver for 2·5 h, the distribution of radioactivity within extracts of bile, blood and liver was determined. The nature of the radioactive products of meta-bolism was inferred by comparison of the distribution of radioactivity after injecting carbon and tritium labelled standards, and by thin-layer chromatography, gas-liquid chromatography, ultraviolet and bioassay analysis.

3. A single injection of 1-¹⁴C PGE₁ indicated that the liver could efficiently remove 89-95% of circulating PGE₁ on a single passage. Biliary excretion was excluded as a major route for elimination of unchanged PGE₁, because only 0·3-0·8% of the injected radioactivity was detected in the bile. During recirculation of 1-¹⁴C PGE₁, 11-19% of the injected radioactivity was detected as exchanged ¹⁴CO₂. The radioactivity detected within liver was identified with further fragments resulting from decarboxylation of PGE₁, which were incorporated into fatty acids and then phospholipids.

4. Studies with 5,6-³H PGE₁, and comparison with the results obtained using 1-¹⁴C PGE₁, revealed a 30-fold increase in the percentage of radioactivity excreted into the bile, suggesting that biliary excretion may be a major route for elimination of compounds smaller than C20 prostaglandin. Evidence that the cyclopentane ring was intact was inferred by formation of a PGB compound on treatment with alkali; similar biliary excretion of 9-³H PGF_1α also occurred. In addition, the increased radioactivity detected within the liver (37%) and blood (43%) after a single injection of 5,6-³H PGE₁ had the solvent partition and thin-layer chromatography properties of PGE₁, but were associated with a less polar compound smaller than the C20 parent structure.

5. These results indicate rapid uptake of circulating prostaglandins by the rat liver. Decarboxylation of prostaglandins results in pharmacological inactivation. The products are excreted into the bile and venous effluent. These processes would curtail the duration of effects following prostaglandin injection.In addition, we infer from these results that any physiological action of these ubiquitous endogenous substances is likely to be localized within their tissue of origin.

     

Age-dependent propranolol clearance in perfused rat liver

The effect of age on the hepatic clearance of propranolol was studied by perfusing the liver isolated from 3- to 104-week-old rats. Propranolol levels in the recirculating perfusate declined biexponentially with time in all age groups. When the liver isolated from 7-week-old rats was perfused with propranolol (1 microgram/ml, 100 ml), hepatic clearance of this drug by the perfused liver (CLperf) increased from 0.589 to 1.14 ml X min-1 X (g liver)-1 with the increase of the perfusion flow rate from 1.0 to 2.0 ml X min-1 X (g liver)-1, confirming evidence of "perfusion-limited" hepatic clearance for this drug. Furthermore, there was no initial concentration(dose)-dependence in CLperf up to 2.5 micrograms/ml (i.e. 250 micrograms/organ). The effect of age on CLperf was then investigated by perfusing the isolated liver with 1.0 micrograms/ml propranolol at 2.0 ml X min-1 X (g liver)-1. Elimination of this drug from the perfusion medium was relatively rapid in 5- to 7-week-old rats, yielding the highest CLperf in these relatively young rats [approximately 1.0 to 1.1 ml X min-1 X (g liver)-1]. In contrast, CLperf values in both immature and older rats were 0.5 ml X min-1 X (g liver)-1 or less. The in vitro intrinsic hepatic clearance estimated in 5- and 7-week-old rats was about ten times as high as that in 104-week-old rats.     

香烟烟雾溶液作用下大鼠淋巴细胞的氧化应激反应

目的　以细胞内活性氧自由基 (ROS)水平、DNA损伤、脂质过氧化物 (LPO)水平以及超氧化物歧化酶 (SOD)活力为指标 ,研究细胞在香烟烟雾溶液作用下产生的氧化应激反应。方法　以PBS作吸收液 ,用大包氏管收集香烟主流烟雾 ,将香烟烟雾溶液 (CSS)分别以 0、1× 10 - 3、2× 10 - 3、4× 10 - 3、8× 10 - 3、12× 10 - 3、16× 10 - 3支 ml的浓度作用于大鼠淋巴细胞。用 2′ ,7′ 二乙酰二氯荧光素 (DCFH DA)测定细胞内ROS水平 ,用彗星试验检测细胞DNA损伤 ,同时检测细胞内SOD活力和LPO水平。结果　 2× 10 - 3支 ml以上剂量组二氯荧光素 (DCF)荧光强度、彗星尾长以及LPO水平均显著高于对照组 ,且随剂量增加而增加。 16× 10 - 3支 ml组SOD活力显著低于对照组。与对照组相比 ,1× 10 - 3支 ml组LPO水平显著降低、SOD活力则显著增高。结论　CSS达到一定剂量时使细胞内ROS水平增高 ,引起细胞DNA损伤和脂质过氧化 ,高剂量时SOD活力降低 ,而低剂量时能够诱导SOD活力