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%.

Monocrotaline pyrrole induces pulmonary endothelial damage through binding to and release from erythrocytes in lung during venous blood reoxygenation.

Monocrotaline has been widely used to establish an animal model of pulmonary hypertension, most frequently in rats. An important feature of this model resides in the selectivity of monocrotaline injury toward the pulmonary vascular endothelium versus the systemic vasculature when administrated at standard dosage. The toxic metabolite of monocrotaline, monocrotaline pyrrole, is transported by erythrocytes. This study aimed to reveal whether partial pressure of oxygen of blood determined the binding and release of monocrotaline pyrrole from erythrocytes in rats with one subcutaneous injection of monocrotatline at the standard dosage of 60 mg/kg. Our experiments demonstrated that monocrotaline pyrrole bound to and released from erythrocytes at the physiological levels of partial pressure of oxygen in venous and arterial blood, respectively, and then aggregated on pulmonary artery endothelial cells. Monocrotaline pyrrole-induced damage of endothelial cells was also dependent on partial pressure of oxygen. In conclusion, our results demonstrate the importance of oxygen partial pressure on monocrotaline pyrrole binding to erythrocytes and on aggregation and injury of pulmonary endothelial cells. We suggest that these mechanisms contribute to pulmonary selectivity of this toxic injury model of pulmonary hypertension.

Pharmacokinetics and Bioavailability Study of Monocrotaline in Mouse Blood by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND AND AIMS: The present study aimed to develop a simple and sensitive method for quantitative determination of monocrotaline (MCT) in mouse blood employing ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI/MS/MS) using rhynchophylline as an internal standard. METHODS: Proteins present in the blood samples were precipitated using acetonitrile. MCT was separated using a 1.7-µm ethylene bridged hybrid (BEH) C18 column (2.1 mm × 50 mm) with a gradient elution program and a constant flow rate of 0.4 mL/min. The LC mobile phase consisted of 10 mmol/L ammonium acetate (containing 0.1% formic acid) and acetonitrile. The total elution time was 4.0 min. The analytes were detected on a UPLC-ESI mass spectrometer in multiple reaction monitoring (MRM) mode and quantified. RESULTS: The new method for the determination of MCT has a satisfactory linear detection range of 1-2000 ng/mL and excellent linearity (r = 0.9971). The lower limit of quantification (LLOQ) of MCT is 1.0 ng/mL. Intra- and interassay precisions of MCT were ≤13% with an accuracy from 96.2% to 106.6%. The average recovery of the new method was >75.0%, and matrix effects were between 89.0% and 94.3%. Based on the pharmacokinetics data, the bioavailability of MCT in mice was 88.3% after oral administration. CONCLUSIONS: The results suggest that the newly standardized method for quantitative determination of MCT in whole blood is fast, reliable, specific, sensitive, and suitable for pharmacokinetic studies of MCT after intravenous or intragastric administration.

7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

Feeding on Host Plants with Different Concentrations and Structures of Pyrrolizidine Alkaloids Impacts the Chemical-Defense Effectiveness of a Specialist Herbivore.

Sequestration of chemical defenses from host plants is a strategy widely used by herbivorous insects to avoid predation. Larvae of the arctiine moth Utetheisa ornatrix feeding on unripe seeds and leaves of many species of Crotalaria (Leguminosae) sequester N-oxides of pyrrolizidine alkaloids (PAs) from these host plants, and transfer them to adults through the pupal stage. PAs confer protection against predation on all life stages of U. ornatrix. As U. ornatrix also uses other Crotalaria species as host plants, we evaluated whether the PA chemical defense against predation is independent of host plant use. We fed larvae from hatching to pupation with either leaves or seeds of one of eight Crotalaria species (C. incana, C. juncea, C. micans, C. ochroleuca, C. pallida, C. paulina, C. spectabilis, and C. vitellina), and tested if adults were preyed upon or released by the orb-weaving spider Nephila clavipes. We found that the protection against the spider was more effective in adults whose larvae fed on seeds, which had a higher PA concentration than leaves. The exceptions were adults from larvae fed on C. paulina, C. spectabilis and C. vitellina leaves, which showed high PA concentrations. With respect to the PA profile, we describe for the first time insect-PAs in U. ornatrix. These PAs, biosynthesized from the necine base retronecine of plant origin, or monocrotaline- and senecionine-type PAs sequestered from host plants, were equally active in moth chemical defense, in a dose-dependent manner. These results are also partially explained by host plant phylogeny, since PAs of the host plants do have a phylogenetic signal (clades with high and low PA concentrations in leaves) which is reflected in the adult defense.

Metabolic activation of retronecine and retronecine N-oxide - formation of DHP-derived DNA adducts.

We have previously reported that metabolism of a series of pyrrolizidine alkaloids in vitro and in vivo generated a set of (+/-)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. It has also been shown that the levels of the DHP-derived DNA adduct formation correlated closely with the tumorigenic potencies of the mice fed with different doses of riddelliine. Retronecine is the necine base and the structurally smallest chemical of the retronecine-type pyrrolizidine alkaloids. Although it has been reported that microsomal metabolism of retronecine generated DHP as a metabolite, it was yet not known whether metabolism of retronecine in vivo could generate DHP-derived DNA adducts and if formed, whether or not the levels of DNA adducts were comparable with those formed from the other tumorigenic retronecine-type pyrrolizidine alkaloids, such as riddelliine, retrorsine, and monocrotaline. In this investigation, the in-vitro and in-vivo metabolic activation of retronecine was studied. Rat liver microsomal metabolism of retronecine in the presence of calf thymus DNA resulted in the formation of a set of DHP-DNA adducts. The metabolism of retronecine N-oxide under similar conditions also formed the similar set of DHP-DNA adducts. The level of DNA adducts from retronecine was enhanced when metabolism by liver microsomes from phenobarbital (PB)-induced rats were used. The DHP-DNA adducts were also found in the liver DNA of female F344 rats treated with retronecine or retronecine N-oxide. The highest level of the total DHP-DNA adducts was found in liver DNA from the rats treated with dehydroretronecine (DHR). The order of the levels of DNA adducts in the liver DNA samples from rats treated with various pyrrolizidine alkaloids was: DHR > riddelliine > riddelliine N-oxide >> retronecine > retronecine N-oxide. The results indicate that 1) retronecine can be metabolized to form DHP by rat liver microsomal enzymes and interacts with DNA to produce DHP-DNA adducts and 2) retronecine N-oxide undergoes the biotransformation to the parent compound, retronecine. The results from this and our previous findings strongly suggest that formation of DHP-DNA adducts may be a potential biomarker for pyrrolizidine alkaloid carcinogenesis.

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo.

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.

Association of dehydromonocrotaline with rat red blood cells.

The association of radiolabeled monocrotaline pyrrole (DHM) with red blood cell (RBCs) ghosts, globins, and heme was examined to determine their role in the transport and stabilization of this hepatic produced putative toxic metabolite of the pyrrolizidine alkaloid monocrotaline (MCT). Rats were administered 5 mg of DHM/kg, i.v., and RBCs and plasma were harvested at 4 and 24 h. Extensive washing of the RBCs with isotonic phosphate buffer did not decrease the amount of radioactivity associated with the cells. The level of DHM equivalents recovered in the RBCs did not decrease between 4 and 24 h, while the plasma levels, which were 29- and 75-fold lower, respectively, decreased from 5.0 to 2.2 nmol of DHM equiv/g of plasma. Globin chains were found to contain 383 and 453 pmol of DHM equiv/mg of protein, respectively. Rats receiving 10 mg of DHM/kg, i.v., with RBCs collected at 2 h, had approximately double the level of radioactivity associated with their RBCs in addition to 2 times the amount of adducts on the globin chains. Globins and ghosts plus heme (2 h) contained 69% and 2% of the radioactivity, respectively. Globin chains treated with an acidic ethanol solution containing AgNO3 resulted in the removal of 31% of the associated radioactivity. GC/ MS and TLC separation of AgNO3-displaced material revealed the presence of the ethyl ether derivatives of 7-hydroxy-1-(hydroxymethyl)-6,7-dihydro-5H-pyrrolizine. The HPLC separation of globin chains revealed that the majority of radioactivity coeluted with the beta-chains. In conclusion, this study found that the administration of radiolabeled DHM resulted in extensive radioactive labeling of RBCs; similar findings have been reported for [14C]MCT.

Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: the role of glutathione and relevance to hepatic venoocclusive disease.

The mechanisms leading to hepatic venoocclusive disease (HVOD) remain largely unknown. Azathioprine and monocrotaline were studied as part of a series of studies looking at a variety of toxins that induce HVOD to find common features that might be of pathogenic significance. In a previous study, dacarbazine showed selective in vitro toxicity to sinusoidal endothelial cells (SEC) compared with hepatocytes and a key role for SEC glutathione (GSH) was demonstrated. Murine SEC and hepatocytes were isolated and studied in culture. Azathioprine and monocrotaline were found to be selectively more toxic to SEC than to hepatocytes. The relative resistance of hepatocytes to azathioprine was due to enhanced GSH defense: hepatocytes exposed to azathioprine maintained intracellular GSH levels better than SEC, particularly when supplemental GSH precursors were added, and hepatocyte resistance was completely overcome by depletion of intracellular GSH. In contrast, monocrotaline toxicity in hepatocytes was largely unaffected by depletion of GSH, which suggests that selectivity of monocrotaline for SEC may be attributable to differences in metabolic activation. Both compounds are detoxified by GSH in SEC, as demonstrated by enhanced toxicity in the presence of buthionine sulfoximine (BSO) and attenuation of toxicity with exogenous GSH. SEC GSH levels were more than 70% to 80% depleted by monocrotaline and azathioprine, respectively, before cell death. Azathioprine and monocrotaline are selectively toxic to SEC; the mechanism of toxicity in the SEC may be caused by profound GSH depletion.

Role of metabolism in monocrotaline-induced immunotoxicity in C57BL/6 mice.

Monocrotaline (MCT) is a pyrrolizidine alkaloid which has been shown to induce immunotoxicity in mice. We hypothesized that metabolic activation of MCT by mixed-function oxygenases (MFO) to dehydromonocrotaline (MCTP) is a prerequisite for its immunotoxicity, as has been shown for other toxic effects of MCT. To test this hypothesis, we compared the in vitro immunotoxic potency of MCT and MCTP to suppress the in vitro antibody response to SRBC and the blastogenic response to B and T cell mitogens. In addition, the effects of in vivo modulation of MFO activities on the immunotoxicity of MCT was examined using phenobarbital (PB) to increase and chloramphenicol (CP) to decrease MCTP production. Results showed that in vitro exposure of splenic lymphocytes to MCT or MCTP produced significant suppression of the antibody and blastogenic responses. MCTP was 200-400-fold more potent than MCT. No metabolism of MCT by splenic cells was detectable, suggesting that unmetabolized MCT is capable of inducing immunotoxicity. In vivo studies showed that, while treatment of mice with PB or CP produced significantly increased and decreased MCTP production by liver microsomes, neither PB or CP treatment significantly altered the immunotoxic potency of MCT. Thus, while the MCTP metabolite is directly immunotoxic in vitro and much more potent than MCT, a role for the MCTP metabolite in MCT immunotoxicity in vivo could not be demonstrated.