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1.
L Nigra  R J Huxtable 《Toxicon》1992,30(10):1195-1202
We have examined the relationship between the metabolism of the pyrrolizidine alkaloid, monocrotaline, and glutathione concentration in the isolated, perfused rat liver. On perfusion of monocrotaline (300 microM) through the isolated liver, high concentrations (1.1 mM) of its metabolite glutathionyldehydroretronecine are released into bile, while much lower amounts (4.86 microM; 0.05 mumol/g liver) accumulate in the perfusate over a 1 hr perfusion period. Metabolite concentration in both the bile and perfusate increase when the level of monocrotaline perfused is increased to 900 microM. Metabolite release is also elevated in livers pretreated with phenobarbital. Monocrotaline perfusion lowered glutathione concentrations in the liver from 30 min onwards. Livers from animals treated with buthionine sulfoximine or chloroethanol showed much lower glutathione levels after 60 min perfusion. Livers from chloroethanol-treated (but not buthionine sulfoximine-treated) animals showed significantly lower release of pyrroles into the bile on perfusion with monocrotaline, but there is no effect on the rate of build-up of pyrrolic metabolites in the perfusate. We conclude that hepatic glutathione concentrations and the release of pyrrolic metabolites of monocrotaline mutually interact. Exposure of the liver to monocrotaline reduces glutathione concentrations, while marked depletion of liver glutathione concentration leads to a decrease in the release of monocrotaline metabolites.  相似文献   

2.
A pneumotoxic pyrrolic metabolite, previously isolated from the bile when rat liver was perfused with the pyrrolizidine alkaloid, monocrotaline, has been identified as 7-glutathionyl-dehydroretronecine. The metabolite showed a TLC spot and HPLC peak corresponding with the latter compound, and a procedure for replacing the thioether group with an ethoxy group converted the metabolite to dehydroretronecine 7-ethyl ether, confirming that the glutathionyl moiety was attached to the 7-position of dehydroretronecine. The same metabolite was detected in bile from rat liver perfused with retrorsine, which is a diester alkaloid similar to monocrotaline, whereas it was not formed from heliotrine, an alkaloid lacking the 7-ester function.  相似文献   

3.
Monocrotaline is a pyrrolizidine alkaloid obtained from the seeds of Crotalaria spectabilis. When perfused through an isolated liver, monocrotaline is metabolized to Ehrlich reactive (E+) metabolites. Metabolism of monocrotaline was faster in livers from male rats than female rats, was inducible with phenobarbital pretreatment, and was inhibited by coperfusion with the P-450 mixed-function oxidase inhibitor SKF-525A, anoxic perfusion conditions, and low temperatures. When metabolites generated by an isolated liver were perfused through isolated lungs in a recirculatory manner, serotonin transport by the pulmonary endothelium was reduced in correlation with the amount of E+ material contained in the perfusion medium. When metabolism of monocrotaline by the liver was inhibited with SKF-525A, low temperature perfusions or anoxic conditions, serotonin transport by the pulmonary endothelium was unchanged from controls. Monocrotaline alone had no effect on the lung. Thus, isolated perfused livers metabolized monocrotaline to chemical species which produced pulmonary damage in vitro. This provides direct evidence that liver metabolites can cause one of the pneumotoxic effects of monocrotaline observed in vivo.  相似文献   

4.
A specific HPLC method with UV detection was used to investigate the disposition of morphine and its metabolites in the in-situ rat isolated perfused liver preparation. Livers of male Sprague-Dawley rats (n = 4) were perfused under single pass conditions with protein-and erythrocyte-free perfusate, containing 2·66 μm morphine, for up to 90 min. The concentration of morphine, normorphine and morphine-3-glucuronide (M3G) in outflow perfusate, and the biliary excretion of M3G and normorphine glucuronide, all reached steady-state levels within 15–20 min after commencing perfusion. At steady-state, the mean (± s.d.) extraction ratio of morphine was 0·87 ± 0·06 and clearance (26·0 ± 1·7 mL min?1) approached perfusate flow rate (30 mL min?1). Although M3G was the main metabolite, accounting for 72·8 ± 12·7% of eliminated morphine, a significant proportion (21·6 ± 13·5%) was N-demethylated to normorphine and was recovered as unchanged normorphine in outflow perfusate and normorphine glucuronide in bile. The biliary extraction ratio of hepatically-formed M3G was 0·61 ± 0·31. Results from an additional six experiments, in which livers were perfused with 1·33 and 2·66 μm of morphine for 30 min each in a balanced cross-over manner, indicated that the disposition of morphine and its metabolites was approximately linear within this concentration range.  相似文献   

5.
Isolated perfused rat liver preparations were utilized to measure the hepatic uptake, biliary excretion and metabolism of rubratoxin B. Livers were perfused with 30% rat blood perfusate containing 0.24 μmol labeled rubratoxin B, and a series of timed blood and bile samples were analyzed by high-pressure liquid chromatography, and treated enzymatically for the determination of glucuronide and sulfate conjugates. Blood, bile and liver samples were also radioassayed. Rubratoxin B was removed from the perfusate by a first-order process (monophasically) with a half-life of 207.5 ± 23.7 min (mean ± SE). By 3.5 hr of perfusion, 30% of the total rubratoxin B-derived radioactivity was excreted into the bile. More than 8% of the total dose of rubratoxin B was excreted unchanged into the bile by 3.5 hr. The rates of biliary excretion of rubratoxin B- derived radioactivity and parent compound reached a maximum at 30 min, after which time the rates of excretion decreased monophasically with half-lives of 35.5 and 72 min, respectively. Two major metabolites detected in the bile were the glucuronide and sulfate conjugates, together accounting for 22% of the radioactivity excreted into the bile by 3.5 hr. In addition, at least one major unidentified organosoluble metabolite was detected in the bile.  相似文献   

6.
Roxithromycin is a macrolide antibiotic with high clinical potency. N-Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver. After addition of roxithromycin (30 μM) to the perfusion medium the parent compound and one major metabolite were detected in bile by high-performance liquid chromatography. The metabolite was identified as monodesmethylated roxithromycin by mass spectrometric analysis. Onset of biliary excretion of native roxithromycin was fast, reaching a maximum (130.52 ±43.88 pmol g?1 min?1) after only 10 min, whereas excretion of the metabolite was delayed (maximum 75.83 ± 11.92 pmol g?1 min?1 at 30 min). The cumulative excretion of roxithromycin and its metabolite into bile during the 60 min of application amounted to only 1.09 ± 0.30 and 0.64 ± 0.22% of the roxithromycin cleared from the perfusate during the same time. The liver content was 0.48 μmol (g liver)?1, indicating high retention within the organ. No release of the metabolite into the perfusate was detected. In conclusion, this study has demonstrated the importance of phase-I metabolism for the biliary excretion of roxithromycin in rat liver. These findings might be predictive of roxithromycin biotransformation and biliary excretion in man.  相似文献   

7.
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.  相似文献   

8.
It has been shown that a short-lived pyrrolic metabolite in fluid flowing out of isolated rat liver perfused with the pyrrolizidine alkaloid, monocrotaline, could be trapped by covalent reaction with a bed of immobilized thiol (thiol-sepharose). Larger amounts of other pyrrolic metabolites, also in the fluid, were not trapped. This provided the first direct support for the widely held hypothesis that reactive pyrrolizidine alkaloid metabolites (dehydro-alkaloids) escape from the liver to damage the lungs of rats in vivo. The relatively smaller proportion of pyrrolic metabolite from retrorsine which could be trapped in this way was consistent with the known lack of pneumotoxicity of this alkaloid. The procedure described should be suitable for trapping other types of electrophilic metabolites.  相似文献   

9.
Wang YP  Yan J  Fu PP  Chou MW 《Toxicology letters》2005,155(3):411-420
Retronecine-based pyrrolizidine alkaloids, such as riddelliine, retrorsine, and monocrotaline, are toxic to domestic livestock and carcinogenic to laboratory rodents. Previous in vitro metabolism studies showed that (+/-)6,7-dihydro-7-hydroxy-1-(hydroxymethyl)-5H-pyrrolizine (DHP) and pyrrolizidine alkaloid N-oxides were the major metabolites of these compounds. DHP is the reactive metabolite of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides are generally regarded as detoxification products. However, a previous study of rat liver microsomal metabolism of riddelliine N-oxide demonstrated that DHP and its parent compound, riddelliine, were generated as the major metabolites of riddelliine N-oxide. In this study the metabolic activation of the three retronecine-based pyrrolizidine alkaloid N-oxides by human liver microsomes is investigated under oxidative and hypoxic conditions. Results shows that both the DHP and the corresponding parent pyrrolizidine alkaloids are the major metabolites of the human liver microsomal metabolism of pyrrolizidine alkaloid N-oxides. Under oxidative conditions, reduction of the N-oxide to pyrrolizidine alkaloid is inhibited and while under hypoxic conditions, DHP formation is dramatically decreased. The oxidative and reductive products generated from the metabolism of pyrrolizidine alkaloid N-oxides are substrate-, enzyme- and time-dependent. In the presence of troleandomycin, a microsomal CYP3A inhibitor, DHP formation is inhibited by more than 70%, while the N-oxide reduction was not affected. The level of microsomal enzyme activity in human liver is comparable with rats. The rate of in vitro metabolism by either human and rat liver microsomes follows the order of riddelliine > or = retrorsine > monocrotaline, and DHP-derived DNA adducts are detected and quantified by 32P-postlabeling/HPLC analysis. Similar DHP-derived DNA adducts are found in liver DNA of F344 rats gavaged with the pyrrolizidine alkaloid N-oxides (1.0 mg/kg). The levels of in vivo DHP-DNA adduct formation is correlated with the level of in vitro DHP formation. Our results indicate that pyrrolizidine alkaloid N-oxides may be hepatocarcinogenic to rats through a genotoxic mechanism via the conversion of the N-oxides to their corresponding parent pyrrolizidine alkaloids, and these results may be relevant to humans.  相似文献   

10.
Severe hepatotoxicity following intoxication with pyrrolizidine alkaloids has been observed in most domestic and laboratory animals. The guinea pig has been an unexplained exception to this generalization. Administration of monocrotaline, a pyrrolizidine alkaloid contained in the seed and vegetation of Crotalaria spectabilis, produced no clinical or pathologic alterations in guinea pigs. The in vitro microsomal ability to convert monocrotaline to its N-oxide derivative was present to the same degree in rats and guinea pigs, but the level of dehydrogenation activity in the rat was considerably greater than in the guinea pig. The ability to produce monocrotaline pyrroles in vivo was also much greater in rats than guinea pigs. However, when the pyrrolic metabolites were administered by iv injection into the mesenteric blood supply, both species developed marked hepatic necrosis. Therefore, monocrotaline N-oxides are not converted to toxic metabolites and are not toxic per se. Monocrotaline pyrroles appear to be the only metabolites yet determined which are responsible for the lesions produced by monocrotaline intoxication. The fact that N-oxidase activity, but not dehydrogenase activity, is present in guinea pig hepatic microsomes supports the contention that N-oxides and pyrroles are formed by separate enzymatic pathways during pyrrolizidine poisoning.  相似文献   

11.
The metabolism and biliary excretion of [14C]phenytoin (DPH) were examined in isolated perfused livers taken from Sprague-Dawley rats pretreated with 0.01, 0.05, 0.1, 0.5, and 1.0 mg/day diethylstilbestrol (DES) sc for 6 days. No difference was seen in the rate of disappearance of DPH from the perfusate or in the perfusate levels of its hydroxylated metabolite, 5-phenyl-5-para-hydroxyphenylhydantoin (HPPH). The biliary excretion of HPPH-glucuronide, however, was significantly depressed in livers from DES-treated rats and resulted in a significant increase in the amount of HPPH-glucuronide appearing in the perfusate. A linear relationship existed between the percentage decrease in biliary excretion of HPPH-glucuronide and the log of the pretreatment dose of DES. Bile flow was significantly depressed at all pretreatment doses of DES such that bile flow was 53.7 and 10.9% of bile flow in controls after 0.01 and 1.0 mg/day DES, respecively. The low bile flow appeared to limit secretion of HPPH-glucuronide in the bile since the maximal concentration of HPPH-glucuronide in bile was greater in livers from DES-treated rats than controls and no significant differences were found in the maximal bile/perfusate concentration ratios of HPPH-glucuronide.  相似文献   

12.
The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 µg ml?1 RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.  相似文献   

13.
The effect of phenobarbital (PB) pretreatment on the biliary excretion of methadone in rats was studied. Possible mechanisms by which PB pretreatment altered the biliary excretion of methadone were considered and studies in vitro on the metabolism of methadone were correlated with findings in vivo. For the biliary excretion studies, 14C-methadone was administered intravenously and biliary excretion measured in anesthetized renal-ligated rats in which the common bile duct was cannulated. PB pretreatment increased the biliary excretion of 14C after 14C-methadone administration. The different metabolites of methadone formed in vivo and excreted into bile were separated by thin-layer chromatography and quantitated. The biliary excretion of the metabolite which results from N-demethylation and cyclization of methadone was not altered by PB pretreatment. However, the biliary excretion of metabolites which result from further N-demethylation, hydroxylation and glucuronidation was increased by PB pretreatment. Several determinants of biliary excretion (i.e. bile flow, hepatic blood flow and metabolism). which are enhanced by PB pretreatment, could cause the observed increase in the biliary excretion of methadone. Of these possibilities, we feel the data best support the suggestion that enhancement of methadone metabolism by PB pretreatment is responsible for the increased biliary excretion of methadone in PB-pretreated rats. Furthermore, metabolism studies in vitro, using microsomes from PB-treated rats, support the suggestion that PB pretreatment enhances the metabolism of methadone in vivo.  相似文献   

14.
The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 microg ml(-1) RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.  相似文献   

15.
Isolated perfused rat liver preparations were utilized to study the drug-induced modification of biliary excretion of [14C]4-chlorobiphenyl (1-CB) and [14C]2,4,5-2′,4′,5′-hexachlorobiphenyl (6-CB) and their metabolites. The effect of pretreatment of rats with mirex was compared with that of phenobarbital (PB) and 6-CB by measuring the biliary excretion of 1-CB and the pharmacokinetics of 1-CB and its metabolites in the perfusate of liver preparations obtained from control and treated rats. The biliary excretion of 1-CB metabolites by mirex-pretreated livers was suppressed by 92% of that in control livers. The rate of metabolism of 1-CB by mirexpretreated livers was slightly decreased. However, the suppression of biliary excretion of 1-CB metabolites does not appear to be related either to decreased rate of metabolism or to the rate of bile flow. These conclusions are borne out from the following two observations: first, the metabolites of 1-CB accumulate at increasing concentrations in the perfusate of mirexpretreated livers; secondly, mirex-pretreated livers had elevated bile flows. Biliary excretion of exogenously added metabolites of 1-CB by mirextreated livers was suppressed by 85% of that of control livers. PB and 6-CB pretreatment caused a statistically significant increase in the biliary excretion of 1-CB metabolites. This was accompanied by a slight but statistically nonsignificant increase in the rate of bile flow. Biliary excretion of 6-CB was completely suppressed by mirex pretreatment and was unaffected by PB. These results indicate that while PB-induced modification of biliary excretion of PCBs may be associated with their metabolism, mirex-induced suppression is associated with the transport of otherwise readily excretable metabolites from the hepatocytes into the bile canaliculi.  相似文献   

16.
The comparative metabolism of the pyrrolizidine alkaloid, [14C]monocrotaline, was studied using rat and guinea pig hepatic microsomes. Metabolites were quantified to the nanomole level using HPLC and radiometric detection. Triorthocresylphosphate and carbon monoxide were used to assess the involvement of carboxylesterases and cytochrome P-450 in the hepatic microsomal metabolism of monocrotaline, respectively. Esterase hydrolysis accounted for 92% of the metabolism in the guinea pig; the rat displayed no esterase activity. This result may explain the guinea pig's resistance to pyrrolizidine alkaloid toxicity. Dehydropyrrole was found to be the major pyrrolic metabolite in the guinea pig, although colorimetric analysis indicated multiple pyrrolic moieties in the rat microsomal incubations.  相似文献   

17.
Monocrotaline (MCT) is a naturally occurring hepatotoxic pyrrolizidine alkaloid found in plants. This investigation is aimed at furthering the understanding of the role of blood in mediating the transport of MCT and its reactive metabolites in humans. Reactions of monocrotaline and its metabolites, dehydromonocrotaline (DHM), retronecine (RET) and dehydroretronecine (DHR) with human blood plasma, red blood cells (RBCs), and whole blood were studied in vitro by proton nuclear magnetic resonance spectroscopy. In plasma MCT remained intact and weakly associated with plasma proteins, and DHM was rapidly hydrolyzed releasing necic and lactone acids, and the reactive pyrrolic metabolite. MCT and its metabolite DHM were internalized in RBCs to the extent of 46.0% and 48.9% respectively in 30 min. No polymerization of DHR was observed when incubated with plasma and RBCs. The data clearly showed that both human plasma and RBCs could be the carriers for the transportation of MCT and its metabolites, DHM, RET and DHR between organs and could stabilise the reactive MCT metabolite DHR.  相似文献   

18.
A reliable technique for rainbow trout liver perfusion has been developed for studies on xenobiotic biotransformation. Normal function of the perfused liver was indicated throughout the perfusion experiment by 1) a proper oxygen consumption, 2) a low leakage of intracellular enzymes, 3) a stable pH value in the effluent, and 4) a stable release of metabolites into the effluent perfusate and bile for at least 2 hr after maximal rate of metabolism was attained. The main metabolite of 7-ethoxycoumarin in effluent perfusate was identified as 7-hydroxycoumarin glucuronide. Only trace amounts were identified as sulfates. When fish were pretreated with Clophen A50 or beta-naphthoflavone, the amount of metabolites released into the effluent perfusate increased 3.4- and 6.4-fold, respectively, when compared to control livers. Furthermore, in livers from Clophen A50- or beta-naphthoflavone-treated fish, only 80 and 67%, respectively, of excreted products were conjugated. Influence of temperature on 7-ethoxycoumarin metabolism was studied in perfused liver and isolated liver microsomes. Results indicate that the Q10 for the metabolism of 7-ethoxycoumarin in perfused liver deviates from that found in isolated microsomes. The amount of metabolites excreted into the bile consisted of about 25% of the amount found in effluent perfusate. The only metabolite detected in bile from perfused liver from control as well as treated fish was 7-hydroxycoumarin glucuronide.  相似文献   

19.
The formation and biliary excretion of the glutathione and cysteine S-conjugates of hexachlorobutadiene were studied in the isolated perfused rat liver. Infusion of increasing amounts of hexachlorobutadiene led to an increase in total metabolite excretion. Partitioning of glutathione conjugate release between bile and perfusate depended on the rate of substrate infusion: S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBG) appeared quantitatively in bile at low hexachlorobutadiene infusion rates, and increasing amounts of the glutathione conjugate were found in the perfusate as infusion rates were increased. The cysteine S-conjugate, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, was not detected in the perfusate, but the amounts found in the bile were correlated with the concentrations of the glutathione conjugate. Depletion of hepatic glutathione concentrations decreased PCBG formation. Hence, at moderate hexachlorobutadiene infusion rates, PCBG is exclusively excreted into bile, indicating that intestinal absorption of PCBG or its metabolites is required for the induction of kidney damage in vivo.  相似文献   

20.
Monocrotaline pyrrole (MCTP) is a reactive metabolite of the pyrrolizidine alkaloid monocrotaline. MCTP given intravenously to rats causes pulmonary hypertension and right ventricular hypertrophy. Lesions in lungs after MCTP treatment contain macrophages and neutrophils, which may contribute to the damage by generation of reactive oxygen metabolites. Rats were treated with MCTP and agents known to protect against oxygen radical-mediated damage in acute models of neutrophil-dependent lung injury. Rats received MCTP and deferoxamine mesylate (DF), dimethyl sulfoxide (DMSO), or polyethylene glycol-coupled catalase (PEG-CAT). MCTP/vehicle-treated controls developed lung injury manifested as increased lung weight, release of lactate dehydrogenase into the airway, and sequestration of 125I-labeled bovine serum albumin in the lungs. Cotreatment of rats with DF, DMSO, or PEG-CAT did not protect against the injury due to MCTP. These results suggest that toxic oxygen metabolites do not play an important role in the pathogenesis of MCTP-induced pulmonary injury.  相似文献   

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