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Administration to rats of monocrotaline pyrrole (MCTP), a putative metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary microvascular thrombosis that is associated with vascular remodeling and pulmonary hypertension. It is possible that vascular thrombi contribute to the lesions that occur after MCTP treatment. Since MCTP is toxic to pulmonary endothelial cells and because the pulmonary endothelium is in a unique position to influence procoagulant and fibrinolytic reactions in the lungs, we examined changes in the procoagulant and fibrinolytic properties of cultured pulmonary artery endothelial cells exposed to MCTP to see if they might favor thrombosis. Monolayers of confluent bovine pulmonary arterial endothelial cells received a single administration of MCTP or N,N-dimethylformamide vehicle, and the media or lysates were examined at 4, 24, 48, or 120 hr after treatment. MCTP caused a time-dependent cell detachment and an increase in release of lactate dehydrogenase into culture medium. Although cell numbers decreased dramatically with time, the protein concentration of cell monolayer lysates was unchanged by treatment. MCTP treatment caused no change in the amount of tissue factor activity/micrograms cellular protein in bovine endothelial cell lysates and only a small increase in the activity of Factor V in the culture medium at 120 hr. In addition, the medium from bovine endothelial cells treated with MCTP had a time-dependent increase in the activity of plasminogen activators and a decrease in the activity of plasminogen activator inhibitors. Thus bovine endothelial cells exposed to MCTP in vitro do not produce changes in these procoagulant or fibrinolytic properties that would explain the thrombosis observed in vivo.

This work has evaluated the clastogenicity of six extracts (tea and aqueous extract of leaves, tea, aqueous and methanolic extracts of dried fruit, and tea of unripe fruit) obtained from Crotalaria retusa L. and three extracts (tea and methanolic extract of dried fruit, and tea of unripe fruit) obtained from Crotalaria mucronata Desv. The extracts were injected intraperitoneally into mice, and the animals were killed 24 h after treatment for preparation of bone marrow cells. The extracts obtained from fruits of Crotalaria retusa were found to cause a dose-dependent increase in the frequency of chromosomal aberrations in mice. On the other hand, no statistically significant increase in the frequency of aberrant cells was observed for the animals treated with leaf extracts obtained from Crotalaria retusa and with extracts from fruits of Crotalaria mucronata. The possibility that the pyrrolizidine alkaloid, monocrotaline, present in Crotalaria retusa exerts a clastogenic effect on mouse bone marrow cells is discussed. Our conclusion is based on studies using intraperitoneal treatments. Effects of oral exposure to extracts of Crotalaria retusa are unknown.

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.

Dietary restriction (DR), i.e. reduction of total caloric intake, has been shown to result in protection against monocrotaline (MCT)-induced pulmonary hypertension (PH). Restriction of the diet to 8 g/rat/day instead of the usual intake (18 g/rat/day), inhibits the progression of cardiopulmonary changes and prolongs survival after a single dose of MCT. We have shown previously that the development of MCT-induced pulmonary hypertension is associated with inhibition of polyamine biosynthesis in the lungs of MCT-treated rats. In the present study, we tested the hypothesis that DR provides protection against the development of chronic PH in the rat by limiting increases in polyamine and DNA synthesis. We randomly divided animals into four groups each (MCT, MCT + DR, control, and control + DR). We injected rats with a single dose of MCT (60 mg/kg, s.c.) and a corresponding number of control rats with vehicle. Animals in all groups were given free access to food and water prior to administration of MCT. Immediately following injection of MCT both the MCT and control groups were given free access to food and water, while the other groups (MCT + DR and control + DR) we given the restricted diet (8 g/rat/day). Daily measurements were made of body weight and of water and food intake. Animals were killed in each group at 1, 4, 7, 14, and 21 days post MCT to determine right ventricular hypertrophy (RVH), lung wet weight, ornithine decarboxylase (ODC) activity, and polyamine and DNA contents. We measured DNA synthesis 7 days after MCT by determining [3H]thymidine incorporation into the whole lung DNA. We found that 7 days after MCT treatment DNA synthesis increased compared to control. However, DR (MCT + DR) treatmen prevented the increase in DNA synthesis following MCT. Right ventricular hypertrophy, lung wet weight, ODC activity and lung polyamine levels were increased following MCT. Treatment with DR (MCT + DR) prevented increases in RVH, lung wet weight, ODC activity and lung polyamine levels. We conclude that DR to 8 g/day/rat protects against MCT-induced PH and is associated with an inhibition of increased lung polyamine and DNA synthesis that occur in the lung during the development of MCT-induced PH. These results are consistent with a recent report which suggests that increased lung polyamine biosynthesis is required for the development of MCT-induced PH. The data are also consistent with the hypothesis that inhibition of polyamine biosynthesis influences the development of MCT-induced PH in part by regulating DNA synthesis in key lung cells.

Administration of monocrotaline pyrrole (MCTP), a putative toxic metabolite of the pyrrolizidine alkaloid, monocrotaline (MCT), results in delayed and progressive pneumotoxicity in the rat. It has been suggested that the lung injury caused by this compound may be initiated by an interaction between MCTP and cells of the pulmonary vasculature. A likely site for initial binding of this reactive electrophile is the pulmonary endothelium. MCTP causes direct toxicity to cultured bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively), but there exist species differences both in whole-animal response to the parent alkaloid and in cellular response to direct application of MCTP. In this study, the changes in cultured rat pulmonary vascular endothelial cells (RECs) after a single administration of MCTP were characterized in order to compare these with changes previously identified in this species in vivo. Studies with RECs have also provided an additional model for examination of species-related differences in response to this toxicant. MCTP caused a delayed and progressive release of lactate dehydrogenase from REC monolayers. Progressive cell detachment was evident and remaining cells became enlarged, with morphologic changes comparable to those reported previously in BECs, including cytoplasmic vacuolization and nuclear enlargement. MCTP inhibited cell proliferation at concentrations of 0.05 micrograms MCTP/ml or greater, and DNA crosslinking was evident at 24 and 48 hr post-treatment. These results suggest that MCTP is directly toxic to cultured RECs, and the development of changes is reminiscent of that seen in the rat in vivo. The cytostatic nature of the compound, in combination with its cytolytic effect on RECs, could contribute to the development of pulmonary edema and other lung vascular changes seen in rats treated with MCT or MCTP.

The pyrrolizidine alkaloid (PA) monocrotaline (MCT) is thought to be activated in the liver to monocrotaline pyrrole (MCTP) which is then transported to the lungs where it causes a pulmonary vascular syndrome characterized by elevated pulmonary artery pressure and right ventricular hypertrophy. We have found that, as opposed to Sprague-Dawley (SD) rats, Fischer 344 (F344) strain rats are resistant to the ventricular hypertrophy and pressure changes induced by MCT. To determine whether this strain difference might be related to differences in hepatic activation of MCT to MCTP, we compared the response of SD and F344 rats to treatment with MCT or MCTP. We determined right ventricular pressure and ventricular weight ratios with each treatment for each strain. We also compared subjective lesion scores of histopathologic changes characteristic of MCT pneumotoxicity. Sprague-Dawley rats treated with either MCT or MCTP had elevated right ventricular pressures (Control 13 +/- 1 mmH2O, MCTP 29 +/- 3 mmH2O, MCT 24 +/- 2 mmH2O) and increased right ventricular weight ratio (RV/LV + S) of 0.30 +/- 0.01 (Control), 0.44 +/- 0.05 (MCTP), 0.44 +/- 0.02 (MCT). Histopathologic evaluation demonstrated that significant alveolar septal fibrosis, edema and type II cell hypertrophy was induced by both PAs in both rat strains but that F344 rats had significantly less vascular medial hypertrophy and adventitial inflammation than SD rats. MCTP treated SD rats had similar vascular and parenchymal alterations as those treated with MCT but with a lesser inflammatory component. We conclude that the strain differences in cardiac and pulmonary vascular responses to MCT also occur with MCTP treatment. This, combined with the similarity in alveolar parenchymal response to both PAs in both strains, suggests that these differences are related to the pulmonary vascular response rather than differences in hepatic metabolism.

Monocrotaline (MCT) produces pulmonary hypertension and right ventricular hypertrophy in rats. It is generally believed that MCT must undergo hepatic metabolism to reactive metabolites that are subsequently transported to the lungs to induce a pneumotoxic response. Several studies suggest that dehydromonocrotaline (MCTP) is the reactive intermediate that initiates pulmonary toxicity. We recently identified two other MCT metabolites, the glutathione and N-acetylcysteine conjugates of 6,7-dihydro-7-hydroxy-1-hydromethyl-5H-pyrrolizine (DHP). To determine the potential pulmonary toxicity of the glutathione conjugate (DHP-GSH) and the unacetylated cysteine conjugate precursor (DHP-Cys) of the N-acetylated excretion product, we conducted parallel in vivo toxicity studies with DHP-GSH, DHP-Cys, MCT, and MCTP. Relative pneumotoxicity was evaluated by measurements of right ventricular pressure (RVP), ventricular weight ratio (RV/LV+S), subjective histopathology, and measurements of components of the arteriolar wall. Animals given a single injection of MCT (60 mg/kg) developed pulmonary hypertension at the end of 3 weeks, as indicated by significant elevation in RVP when compared to the controls (22.1 +/- 2.4 mm Hg vs 13.2 +/- 0.8 mm Hg). A parallel and significant increase in RV/LV+S was also evident: 0.37 +/- 0.021 (MCT) vs 0.299 +/- 0.011 (control). Histopathology showed marked alterations in both pulmonary vasculature and parenchyma in MCT- and MCTP-treated animals. MCTP (1 mg/kg) caused a significantly elevated RVP (MCTP vs control: 28.1 +/- 3.4 mm Hg vs 16.8 +/- 0.97 mm Hg) and an increased RV/LV+S (MCTP vs control: 0.445 +/- 0.051 vs 0.284 +/- 0.026). Both MCT- and MCTP-treated rats had increased arteriolar medial thickness and decreased lumen diameter, but MCTP-treated rats had a milder vascular inflammatory response and less parenchymal lesions. Neither DHP-GSH (24 or 12 mg/kg) nor DHP-Cys (12 mg/kg) caused detectable changes in pulmonary circulation and no structural alteration in the lung was observed in these treatment groups. Although they are all pyrrolic metabolites of MCT, these studies demonstrate that only MCTP but not the glutathione or cysteine conjugates, is pneumotoxic at the doses tested.

The level of clastogenic damage in mice following in vivo adult and transplacental exposure to benzidine, heliotrine, monocrotaline and urethane was compared using the micronucleus (MN) assays of polychromatic erythrocytes (PCE) found in mouse adult bone marrow and foetal liver. Saline was used as a negative control. Swiss-albino mice (gestation 17-19 days) were given a single acute intraperitoneal injection. Animals sacrificed 21 h after 0.75 LD50 of each chemical had significantly increased frequencies of micronucleated PCE in adult and foetal tissues (p < 0.05). Heliotrine resulted in the largest increases in mean value of micronucleated PCE, of 5.4% in adult bone marrow and 6.9% in foetal liver compared to control values of 0.7% and 0.9% for adult and foetal tissue respectively (p < 0.005). The induction of MN was significantly higher in foetal than adult cells for 0.75 LD50 monocrotaline and benzidine after 21 h (p < 0.0005), and for one LD50 heliotrine at 12, 24 and 30 h after injection in a time-response study (p < 0.02). Induction of MN following LD50 heliotrine showed peak MN expression in PCE at 18 h after injection for adult bone marrow (2.6%) and at 24 h for foetal liver (7.9%). The majority of micronucleated PCE in adult and foetal tissue contained only one MN (mean 74% in mice treated with chemicals and 91% in untreated mice). This study demonstrated the sensitivity of the transplacental MN assay for detecting clastogenic damage in mice induced by chemicals and their metabolites. The importance of monitoring indicators of toxicity was highlighted by changes in erythrocyte population composition and the presence of basophilic stippling following exposure to LD50 heliotrine.

Administration of the pyrrolizidine alkaloid monocrotaline (MCT) to rats results in hypertensive pulmonary vascular disease characterized by a structurally based increase in pulmonary vascular resistance and right ventricular hypertrophy. Alterations in lung angiotensin converting enzyme activity in MCT-treated rats have suggested a role for angiotensin II (AII) in the pathogenesis of this model of hypertensive pulmonary vascular disease. To determine if increases in AII contribute to the development of pulmonary hypertension in MCT-treated rats, we examined the effect of chronic administration of the nonpeptide AII receptor antagonist Losartan on indices of pulmonary hypertension, Losartan (DuP 753; 10 mg/kg s.c.) administration for 21 days did not prevent the development of hypertensive pulmonary vascular disease in MCT-treated rats. However, 18 hr after the last dose of Losartan, AII (0.1 micrograms/kg i.v.)-induced pressor responses were inhibited by 63% in Losartan-treated rats. Losartan administration in MCT-treated rats did not prevent increases in pulmonary artery pressure or development of right ventricular hypertrophy. Additionally, increases in medial arterial thickness in pulmonary artery vessels (less than 50 microns and 50-100 microns external diameter) from MCT-treated rats were still evident in Losartan-treated rats. However, Losartan administration decreased medial pulmonary artery thickness of 50 to 100 microns external diameter vessels in control rats. These results demonstrate that AII. acting at the AT1 receptor subtype, does not contribute to pulmonary hypertension in this animal model.

Three pyrrolizidine alkaloids (PAs), monocrotaline, retrorsine and isatidine, were tested for their clastogenic activity under different conditions of metabolic activation in vitro. All three compounds exhibited a weak activity when V79 cells were treated at very high concentrations for 18 h in the absence of a metabolizing system. Short-term (2 h) treatment with rat liver S9 mix led to a strong and concentration-dependent increase in chromosomal aberrations for retrorsine. Isatidine was not mutagenic with S9 mix and monocrotaline was positive at high concentrations only. In contrast, a prolonged treatment (18 h) in vitro under activation conditions in the presence of primary hepatocytes led to clear concentration-dependent positive responses for all three PAs investigated. Particularly the results with isatidine demonstrate that in vitro tests using S9 mix for metabolization can generate misleading results. It is not clear whether the results could be attributed to a better activation of the test compounds by intact hepatocytes in comparison to S9 mix or if the fact that only hepatocytes allow a treatment for the whole culture period under activation conditions was more important. Owing to its strong cytotoxicity the exposure to S9 mix is generally limited to 2-4 h, limiting also the exposure of the target cells to a test chemical as well as its metabolites. The results presented show significant differences in mutagenic potency of PAs due to variations in the activation system. This underlines the usefulness of primary hepatocytes, e.g., for the detection of pre-mutagens. The PAs investigated are present in plants which are used for phytotherapeutic medicinal products. They do not contribute to their efficacy and are, therefore, not to be tolerated in amounts that may impose a risk for the user.

Hepatic venoocclusive disease is a frequent lethal complication of bone marrow transplantation. It has also been associated with hepatic irradiation and administration of chemotherapeutic agents without BMT. The pathogenesis and therapy of VOD are unclear. The present studies were directed at developing a canine model for VOD. Three groups of dogs were studied. Group one consisted of 8 dogs in which monocrotaline (MC) was administered at 125 mg/kg orally on an intermittent schedule. In 7 of the 8 dogs 6 to 9 doses of drug were administered between 42 and 110 days. Group 2 consisted of 6 dogs receiving busulfan 2 mg/kg/day for 17-25 days, when platelet counts decreased to less than 5 x 10(4)/mm3 or clinical bleeding occurred. Group 3 consisted of 2 dogs receiving 24 Gy and 4 dogs receiving 36 Gy of whole-liver irradiation. Seven of 8 dogs in group 1 developed significant liver function abnormalities and evidence of portal hypertension. Histologic findings of VOD were present at autopsy. Group 2 dogs failed to develop clinical or laboratory liver abnormalities, but 3 of 6 animals had minimal histologic evidence of VOD. Three of 6 dogs in group 3 receiving 36 Gy developed hepatic dysfunction and had findings of fibrosis at autopsy. It was concluded that MC administration produced consistent clinical and histologic features of VOD in dogs. Changes occurring after busulfan or total-liver irradiation administration were less reproducible. Dogs are a suitable large-animal model for studies of VOD.

On the basis of the previous findings that alpha-difluoromethylornithine (DFMO, an inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme in polyamine biosynthesis) treatment prevents monocrotaline-(MCT) induced pulmonary hypertension and that ventilatory dysfunction precedes pulmonary hypertension in MCT-treated rats, we hypothesize that MCT-induced changes in airway/lung function are polyamine dependent. To evaluate this hypothesis, in phase 1, 48 young Sprague-Dawley rats were evenly divided into four groups: control, DFMO, MCT, and DFMO + MCT. Each DFMO rat received DFMO in its drinking water (2%) for 11 days, with additional injections (400 mg/kg sc) on the 5th day. Each MCT rat received a single injection of MCT (60 mg/kg sc) 1 wk before the functional study. Each DFMO + MCT rat received the same DFMO and MCT treatments as above, and MCT was administered on the 5th day of the DFMO treatment. In the MCT group, there were marked rightward shifts in pressure-volume and maximal flow-static recoil (MFSR) curves and significant decreases in dynamic and quasi-static compliance, the maximal expiratory flow, slope of the MFSR curve, and the carbon monoxide diffusing capacity, as well as a significant increase in alveolar wall thickness. However, in rats treated with DFMO + MCT, most of MCT-induced changes were significantly attenuated. To evaluate whether MCT causes bronchoconstriction, a bronchodilator, terbutaline (0.2 mg/kg i.v.), was administered to control (n = 7) and MCT (n = 11) rats in phase 2. Terbutaline significantly reversed MCT-induced decreases in maximal expiratory flow and slope of the MFSR curve, whereas it did not alter these parameters in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocrotaline (MCT) is an 11-membered macrocyclic pyrrolizidine alkaloid (PA) that causes a pulmonary vascular syndrome in rats characterized by proliferative pulmonary vasculitis, pulmonary hypertension, and cor pulmonale. Current hypotheses of the pathogenesis of MCT-induced pneumotoxicity suggest that MCT is activated to a reactive metabolite(s) in the liver and is then transported by red blood cells (RBCs) to the lung, where it initiates endothelial injury. While several lines of evidence support the requirement of hepatic metabolism for pneumotoxicity, the mechanism and relative importance of RBC transport remain undetermined. The endothelial injury does not appear to be acute cell death but rather a delayed functional alteration that leads to disease of the pulmonary arterial walls by unknown mechanisms. The selectivity of MCT for the lung, as opposed to that of other primarily hepatotoxic PAs, appears likely to be a consequence of the differences in hepatic metabolism and blood kinetics of MCT. A likely candidate for a reactive metabolite of MCT is the dehydrogenation product monocrotaline pyrrole (MCTP). Secondary or phase II metabolism of MCT through glutathione (GSH) conjugation has been characterized recently and appears to represent a detoxification pathway. The role of inflammation in the progression of MCT-induced pulmonary vascular disease is uncertain. Both perivascular inflammation and platelet activation have been proposed as processes contributing to the response of the vascular media. This review presents the experimental evidence supporting these hypotheses and outlines additional questions that arise from them.

Monocrotaline (MCT)-induced vascular injury in liver and lung may be caused by interaction of MCT metabolites such as monocrotaline pyrrole (MCTP) with vascular cells. Responses of bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively) to a single administration of MCTP were compared. MCTP caused a delayed and progressive release of lactate dehydrogenase (LDH) activity from BECs and a gradual decrease in monolayer cellularity. Surviving cells became markedly hypertrophic. PECs were less sensitive to the cytolytic effects of MCTP, showing minimal cell detachment and little release of LDH activity. However, monolayer cellularity, as assessed by PEC enumeration, decreased in a dose-dependent manner. Hypertrophy of surviving PECs was less pronounced than in BECs. MCTP caused enhanced release of prostacyclin from monolayers of BECs and PECs exposed to 10 micrograms MCTP/ml, and concentrations of 0.5 microgram/ml or greater caused equivalent reduction in colony-forming efficiency in both cell types. In summary, whereas BECs were more susceptible to the cytolytic and hypertrophic effects of MCTP, BECs and PECs responded similarly with regard to prostacyclin release and were equally sensitive to the cytostatic effects of this compound.

Monocrotaline (MCT) is a member of a class of naturally occurring phytotoxins known as pyrrolizidine alkaloids, and is a toxicological concern to both man and his livestock. The purpose of these studies was to evaluate the effect of a 14-day oral MCT (0-100 mg/kg per day) exposure on the functional integrity of various immunocyte effector systems in C57BL/6 mice, as well as to investigate potential mechanisms for its immunotoxicity. Decreases in lymphoid organ weights and cellularity, and resident peritoneal exudate cell (PEC) number were only observed after exposure to the highest dose of 100 mg/kg MCT. This dose also inhibited NK cell cytotoxicity, while the total number of NK lytic units per spleen was decreased (-53%) after exposure to 50 mg/kg MCT. Following i.p. injection of SRBC, the percentage of PEC macrophages containing engulfed SRBC was significantly increased in MCT-exposed mice, while the percentage of large vacuolated (activated) macrophages was decreased in a dose-dependent manner. Exposure to MCT significantly decreased the total number of Ig+ cells without altering the number of CD4+ and CD8+ cells. The antibody responses (PFC/10(6) spleen cells) to two T cell-independent antigens, TNP-LPS and DNP-Ficoll, were significantly decreased at all MCT doses, and the degree of suppression of both responses was identical at coincident doses. MCT exposure (25 mg/kg) significantly suppressed the blastogenic response to the T cell mitogen concanavalin A (-38%), and to the B cell mitogen lipopolysaccharide (-58%). These results indicate that exposure to MCT can alter the functional integrity of various immune effector responses in a dose-dependent manner, and suggest that the B cell may be a relatively more sensitive target of MCT immunotoxicity compared to T cells, macrophages and NK cells.

Six compounds related to pyrrolizidine alkaloids have been subjected to an in vitro mammalian cell transformation test. Two hepatocarcinogenic alkaloids (retrorsine and monocrotaline) and one synthetic analogue (synthanecine A bis-N-ethylcarbamate) gave positive results while a non-toxic alkaloid (rosmarinine) was negative in the test. Positive results were also given by dehydroretronecine, a secondary pyrrolic alkaloid metabolite, and the closely related synthetic compound 2,3-bishydroxymethyl-1-methylpyrrole. These observations lend support to the hypothesis that a simple alkylating pyrrole is the biologically active chemical agent derived from these alkaloids. The negative transformation response observed for the non-carcinogenic alkaloid rosmarinine establishes that the carcinogenic alkaloids are inducing transformation rather than simply selecting for spontaneous transformants. The mammalian-derived cells used in this study, unlike S. typhimurium, were capable of activating retrorsine in the absence of an auxiliary source of metabolising enzymes (S-9 mix).