Simultaneous determination of monocrotaline and its N-oxide metabolite in rat plasma using LC-MS/MS: Application to a pharmacokinetic study.

Monocrotaline (MCT) is a pyrrolizidine alkaloid that can induce hepatic sinusoidal damage, pulmonary hypertension, renal toxicity, and heart disease. Monocrotaline N-oxide (MNO), the primary metabolite of MCT, is less toxic; however, it can convert back to MCT to exhibit its toxicity. This study developed and validated a rapid and sensitive LC-MS/MS method for the simultaneous determination of MCT and monocrotaline N-oxide in rat plasma. The method has a linearity over the concentration range of 1-2000 ng/mL with correlation coefficients (r) >0.997 for each analyte. The results of selectivity, matrix effect, accuracy and precision, and recovery were all within the acceptance criteria. The validated method has been successfully applied to study pharmacokinetic behaviors and bioavailability of MCT in rats. MCT was rapidly absorbed (Tmax : 0.400 ± 0.149 h) after oral administration, and the absolute bioavailability of MCT was 78.2%.

Lung injury induced by pyrrolizidine alkaloids depends on metabolism by hepatic cytochrome P450s and blood transport of reactive metabolites.

Pyrrolizidine alkaloids (PAs) are common phytotoxins with both hepatotoxicity and pneumotoxicity. Hepatic cytochrome P450 enzymes are known to bioactivate PAs into reactive metabolites, which can interact with proteins to form pyrrole-protein adducts and cause intrahepatic cytotoxicity. However, the metabolic and initiation biochemical mechanisms underlying PA-induced pneumotoxicity remain unclear. To investigate the in vivo metabolism basis for PA-induced lung injury, this study used mice with conditional deletion of the cytochrome P450 reductase (Cpr) gene and resultant tissue-selective ablation of microsomal P450 enzyme activities. After oral exposure to monocrotaline (MCT), a pneumotoxic PA widely used to establish animal lung injury models, liver-specific Cpr-null (LCN) mice, but not extrahepatic Cpr-low (xh-CL) mice, had significantly lower level of pyrrole-protein adducts in the serum, liver and lungs compared with wild-type (WT) mice. While MCT-exposed LCN mice had significantly higher blood concentration of intact MCT, compared to MCT-exposed WT or xh-CL mice. Consistent with the MCT in vivo bioactivation data, MCT-induced lung injury, represented by vasculature damage, in WT and xh-CL mice but not LCN mice. Furthermore, reactive metabolites of MCT were confirmed to exist in the blood efflux from the hepatic veins of MCT-exposed rats. Our results provide the first mode-of-action evidence that hepatic P450s are essential for the bioactivation of MCT, and blood circulating reactive metabolites of MCT to the lung causes pneumotoxicity. Collectively, this study presents the scientific basis for the application of MCT in animal lung injury models, and more importantly, warrants public awareness and further investigations of lung diseases associated with exposure to not only MCT but also different PAs.

¹H NMR study of monocrotaline and its metabolites in human blood.

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.

m-Nisoldipine attenuates monocrotaline-induced pulmonary hypertension by suppressing 5-HT/ERK MAPK pathway.

Effect of new calcium antagonist m-nisoldipine (m-Nis) on MCT-induced PH in rats and its mechanisms were investigated. Rats were injected with a single dose (60 mg x kg(-1)) of MCT subcutaneously to induce PH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. PCNA, ERK1 and p-ERK expressions were analyzed by Western blotting. The expressions of 5-HT and PCNA were observed with immunohistochemistry. Results suggested that the PAP, right ventricular index and the degree of muscularization of small pulmonary artery were elevated markedly in MCT group, which was attenuated by m-Nis treatment. A significant reduction in MDA production and an increase in the SOD activity in the serum were also observed in all three m-Nis groups. The number of PCNA and 5-HT positive smooth muscle cells increased significantly in MCT group, and m-Nis treatment attenuated the expression obviously. Western blotting results suggested that the protein expression of PCNA and the ratio of p-ERK/ ERK1 increased markedly in MCT group and decreased by m-Nis. In conclusion, m-Nis protected against MCT-induced PH by decreasing PAP, right ventricular index, PAMSCs proliferation and pulmonary artery remodelling, which may be related to the reduction of 5-HT and the suppression of the ERK/MAPK signal pathway.

Fibrinolytic activity in blood and lungs of rats treated with monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP), a putative toxic metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary vascular thrombi that are associated with vascular remodeling, pulmonary hypertension, and right cardioventricular hypertrophy in rats. It is possible that such thrombi contribute to the lung injury and pulmonary hypertension in this model. A previous study indicated that rats treated with MCTP did not have excessive procoagulant activity in the peripheral blood. Since thrombosis may also result from insufficient fibrinolytic activity in the systemic circulation, we evaluated the fibrinolytic system of rats given MCTP. Male Sprague-Dawley rats were given a single injection of MCTP (3.5 mg/kg) or an equal volume of N,N-dimethylformamide vehicle in the tail vein and were killed at 1, 3, 5, 8, 11, or 14 days after toxin administration. Several markers of lung injury and fibrinolysis were measured. Lung injury was evident 3 days after administration of MCTP and became more pronounced with time. In MCTP-treated rats, right heart hypertrophy was observed at 11 days and became more pronounced at 14 days. There was no change in the plasminogen concentration or in the activities of tissue plasminogen activator or alpha 2-antiplasmin in blood throughout the time course. Beginning at Day 8 and continuing through Day 14, there was an increase in the activity of plasminogen activator inhibitor in blood of rats that received MCTP. In addition, we evaluated the fibrinolytic activity of lung tissue slices. Rats treated with MCTP had a significant decrease in fibrinolytic activity of lung tissue at Day 3. A more pronounced decrease was evident by Day 8, and this continued through Day 14. In summary, MCTP treatment of rats decreases the fibrinolytic activity of lung tissue relatively early after exposure to the toxicant and before the onset of pulmonary hypertension. The change is reflected slightly later in plasma. These alterations in fibrinolytic activity may explain why fibrin thrombi form in the lungs of rats treated with MCTP.