Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells.

The mechanistic relationship between the widely used monocrotaline model of primary pulmonary hypertension and altered TGFbeta family signaling due to genetic defects in the Bone Morphogenetic Protein type II receptor in affected humans has not been investigated. In this study we use fluorescent microscopy to demonstrate nuclear translocation of Smad 4 in human pulmonary arterial endothelial cell (HPAEC) cultures treated with monocrotaline pyrrole (MCTP), Bone Morphogenetic Protein (BMP) and TGFbeta. While MCTP induced transient nuclear accumulation of phosphorylated Smad 1 (P-Smad 1) and phosphorylated Smad 2 (P-Smad 2), only expression of P-Smad 1 was significantly altered in western blots. P-Smad 1 expression significantly increased 30 min following treatment with MCTP correlating with P-Smad 1 and Smad 4 nuclear translocation. Although a modest, but significant decrease in P-Smad 1 expression occurred 1 h after treatment, expression was significantly increased at 72 h. Evaluation of components of the signal and response pathway at 72 h showed decreased expression of the BMP type II receptor (BMPrII), no change in TGFbeta Activin Receptor-like Kinase 1 (Alk 1), no change in Smad 4 but increase in the inhibitory Smad 6, decrease in the alternate BMP signaling pathway p38(MAPK) but no change in the psmad1 response element ID 1. Our results suggest transient activation of Smad signaling pathways in initial MCTP endothelial cell toxicity, and a persistent dysregulation of BMP signaling. Electron microscopy of cell membrane caveoli revealed a dramatic decrease in these structures after 72 h. Loss of these structural elements, noted for their sequestration and inhibition of receptor activity, may contribute to prolonged alterations in BMP signaling.

The BMP type II receptor is located in lipid rafts, including caveolae, of pulmonary endothelium in vivo and in vitro.

Polymorphic mutations in the Bone Morphogenetic Protein type II receptor (BMPrII) gene have been implicated in the development of familial primary pulmonary hypertension (PPH) however, the role BMPrII mutations play in the development of PH has not yet been elucidated. Endothelial caveolae are an important domain of hemodynamics containing eNOS, the serotonin transporter, and endothelin receptors. In this study we show by standard immunohistochemistry (IHC) that BMPrII is widely distributed in the vasculature of the rat lung, and more specifically distributed to both apical and basal membranes of the arteriolar endothelium by fluorescent IHC. We also examined compartmentalization of BMPrII in lipid fractions of plasma membranes isolated by silica based extraction from human pulmonary artery endothelial cells and rat lung endothelium. Density gradient centrifugation demonstrated BMPrII in separate caveolin-1 (cav-1) and non-cav-1 lipid rich fractions. Electron microscopy co-localized cav-1 and BMPrII in flask shaped membrane fragments. Three-dimensional fluorescence microscopy demonstrated BMPrII in discrete membrane foci, a portion of which were co-localized with cav-1, as well as in Golgi. Our findings indicate that BMPrII is located within lipid-dense fractions of pulmonary endothelial cell membranes with a portion present in caveolae suggesting potential dynamic regulatory structural relationships.

Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells.

A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [(14)C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A(3) (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase beta-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nucleophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria.

Characterization of cytosolic glutathione S-transferases in juvenile Chinook salmon (Oncorhynchus tshawytscha).

Four cytosolic glutathione S-transferase (GST) classes were isolated and characterized from juvenile winter run Chinook salmon (Oncorhynchus tshawytscha) liver. Two techniques were used: (1) gel electrophoresis/immunoblotting against a polyclonal striped bass GST antibody and (2) high-pressure liquid chromatography (HPLC). Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to elucidate peptide sequences and the proteins were identified as pi, theta, mu and alpha, by searching against the NCBI non-redundant database (nrDB). Catalytic activity of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA) were determined to be 0.3+/-0.05 U/mg cytosolic protein and 0.06+/-0.02 U/mg cytosolic protein, respectively.

Characterization of glutathione S-transferases in juvenile white sturgeon.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thus preventing toxicity. This study characterized the cytosolic GST classes of juvenile white sturgeon (Acipenser transmontanus) liver, using two methods of isolation. The first, which employed affinity chromatography, electrophoresis and immunoblotting against a polyclonal striped bass GST antibody, yielded two cytosolic GSTs. The GSTs were identified by nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS), peptide mass mapping and MS/MS sequencing, as well as de novo MS/MS sequencing as GST classes pi and mu using the Mascot search engine and the NCBI non-redundant database (nrDB) for both methods. The molecular masses were determined to be 23,548 +/- 23 and 26,027 +/- 23 Da, respectively, using linear matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The second method of isolation, which used affinity chromatography and high-pressure liquid chromatography (HPLC), yielded pi, mu, and possibly two alpha isoforms by MALDI-TOF-TOF, again searching against the NCBI nrDB. The alpha isoforms were determined to have molecular masses of 25,528 +/- 23 and 25,348 +/- 23 Da by electrospray ionization source (ESI)-MS. Overall, it appears that the HPLC method is more sensitive than immunoblotting with the current antibody. Activity of the cytosolic GSTs was evaluated using the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and found to be 2.4 +/- 0.6 U/mg cytosolic protein, and 0.41 +/- 0.05 U/mg cytosolic protein using ethacrynic acid (ETHA).

Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins.

Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell-cell occludens junction molecule (ZO-1), the cell-cell adherens junction linker, ss-catenin, and the cell-matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro.

Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer.

We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene.

DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells.

Monocrotaline pyrrole (MCTP) causes cyto- and karyomegaly and persistent cell cycle arrest in the G2 stage of the cell cycle in cultured bovine pulmonary artery endothelial cells. To better characterize the cell cycle regulatory mechanisms of this process as well as determine whether this process would occur in cells of human origin, we treated human pulmonary artery endothelial cell (HPAEC) cultures with MCTP and determined, by flow cytometry, the expression of cyclin B1 and p53 in conjunction with DNA content. We also validated by Western blots that the persistence of cdc2 in its inactivated phosphorylated state, previously described in bovine cell cultures, occurred in HPAEC. Alterations in p53, cyclin A, cyclin B1, and cdc25c expression were also examined in Western blots of treated HPAEC extracts. The response of HPAEC to MCTP was compared with that of adriamycin and nocodazole, agents known to cause cell cycle alterations. Results of these experiments demonstrate that HPAEC treated with MCTP develop a population of cells in G2 that has increased cyclin B1 expression. These cells express increased amounts of cdc2 but not cdc25c. The ratio of inactive triphosphorylated cdc2 to the active monophosphorylated form increased moderately from control cultures in contrast to predominance of the active form in nocodazole-treated cultures. In addition, a second population of cells expressing cyclin B1 had continued incorporation of BrdU and DNA content consistent with 8 N chromosomes. A similar 8 N cell population was evident in nocodazole-treated cells but these cells had both cyclin B1 positive and negative components. Compared with adriamycin, a known inducer of p53, MCTP-treated HPAEC expressed p53 only at high concentrations and p53 expression was not coordinated with G2 arrest or polyploidy. We conclude that HPAEC treated with low concentrations of MCTP develop G2 arrest in association with persistent cyclin B1 expression, failure to completely activate cdc2, and continued DNA synthesis through a pathway that is unrelated to altered expression of p53.

Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells.

A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for (14)C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), beta- or gamma-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline.

Manipulation of injury and repair of the alveolar epithelium using two pneumotoxicants: 3-methylindole and monocrotaline.

The role of type II epithelial cell proliferation in repair of diffuse alveolar epithelial injury was examined using two pneumotoxicants, 3-methylindole (3-MI) and monocrotaline (MCT). It was hypothesized that if MCT inhibits type II epithelial cell mitosis, then pulmonary fibrosis would result after diffuse 3-MI-induced type I alveolar cell injury in rats preadministered MCT. Four groups of rats were given vehicle control, MCT, 3-MI, or MCT and 3-MI. Lungs from rats killed 4 days post-treatment were examined subjectively and quantitatively by light and electron microscopy. Proliferative stimulus was estimated by bromodeoxyuridine (BrdU) incorporation. Lungs from rats killed 2 weeks post-treatment were evaluated by light microscopy. At 4 days, the number of type II cells in the lungs of 3-MI-treated rats was 3 times greater than in the lungs of the dually (MCT/3-MI) treated rats which was the same as the control rat lungs. There was no significant difference between the MCT/3-MI-treated rats and the 3-MI-treated rats with regard to the percentage of denuded alveolar basement membrane. The number of BrdU-labeled type II epithelial cells was increased above the control in both 3-MI-treated groups, but was greater in the 3-MI-treated rat lungs than in the lungs of the MCT/3-MI-treated rats. The average type II cell volume in dually treated rats was 3 times the volume in the control animals and 50% greater than that in 3-MI-treated rats. Transmission electron microscopy of the lungs of the MCT/3-MI-treated rats demonstrated flattened hypertrophic type II cells over large portions of the basement membrane. The light microscopic appearance and collagen staining of the lungs of the dually treated rats were similar to the negative control rat lungs 2 weeks after dosing with 3-MI. This suggests that despite a proliferative stimulus, MCT inhibits type II cell division after diffuse alveolar type I cell injury, but that type II cell migration and coverage of the basal lamina proceed. Results of this study suggest that coverage of the denuded basal lamina by any method is sufficient to prevent interstitial alveolar fibrosis.

Monocrotaline pyrrole interacts with actin and increases thrombin-mediated permeability in pulmonary artery endothelial cells.

One of the earliest morphologic changes evident in the monocrotaline (MCT) model of pulmonary hypertension in rats is microvascular leak. Whether this represents a direct effect of MCT metabolites or is secondary to inflammatory and thrombotic changes remains uncertain. To determine whether MCT directly affects endothelial cell permeability barrier function, we characterized the interaction of the reactive pyrrole intermediate of MCT (MCTP) with endothelial cell actin and characterized its effects on thrombin-mediated signal transduction and monolayer permeability. Bovine pulmonary endothelial cells (BPAEC) treated with MCTP had altered distribution of filamentous actin evident by fluorescence microscopy. Correlative Western blots and autoradiography of actin isolated from BPAEC treated with 14C-MCTP showed comigration of actin and MCTP-derived 14C. MCTP treatment did not alter cellular free Ca2+ concentrations nor did it interfere with thrombin-mediated intracellular Ca2+ signal. Pretreatment with MCTP significantly augmented the thrombin-mediated transudation of Evan's blue albumin in BPAEC monolayers apparently by increasing the size of intercellular gaps. We conclude that MCTP directly interacts with actin to alter its polymerization state but does not significantly affect endothelial cell response to contractile stimulus. Our results suggest that MCTP may affect endothelial cell barrier function through alterations in intracellular junctions.

Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells.

In the monocrotaline (MCT) model of pulmonary hypertension, the pulmonary vascular endothelium is the likely early target of the reactive metabolite monocrotaline pyrrole (MCTP). Incubation of cultured bovine pulmonary arterial endothelial cells (BPAEC) with MCTP results in covalent binding to DNA, cell cycle arrest, and delayed but progressive cell death. The mode of cell death in MCTP-induced endothelial damage has not been addressed previously. Since DNA damage is frequently associated with apoptosis, the presence or absence of apoptosis in adherent BPAEC was determined by several techniques, including morphologic and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling. Two concentrations of MCTP (5 and 34.5 microgram/ml) along with a vehicle control were examined with each assay. Both concentrations of MCTP induced increasing numbers of cells to undergo apoptosis over time beginning as early as 6 h after exposure to MCTP in the high concentration group. Control and vehicle control cells exhibited small amounts of apoptosis (1-2%), which did not change over the duration of the experiment. Additionally, cell membrane integrity was assessed over time by either exposure to membrane-impermeant dyes or measuring LDH release. By either method, BPAEC had increased membrane permeability after about 48 h of either low or high concentration MCTP exposure. We conclude that both a low or high concentration of MCTP causes cell death in BPAEC by inducing apoptosis.

Prolonged cell-cycle arrest associated with altered cdc2 kinase in monocrotaline pyrrole-treated pulmonary artery endothelial cells.

Monocrotaline pyrrole (MCTP), a metabolite of the pyrrolizidine alkaloid monocrotaline, is thought to initiate damage to pulmonary endothelial cells resulting in delayed but progressive pulmonary interstitial edema, vascular wall remodeling, and increasing pulmonary hypertension. MCTP was previously shown to inhibit pulmonary endothelial cell proliferation and cause cell-cycle arrest in vitro. To determine the persistence of arrest and better characterize the cell-cycle stage at which it occurs, bovine pulmonary artery endothelial cells (BPAEC) under differing growth conditions were exposed to low (5 microg/ml) or high (34.5 microg/ml) concentrations of MCTP for varying times. Flow cytometric cell-cycle analysis was coupled with Western blot and biochemical analysis of cdc2 kinase and measurements of cell size. MCTP treatment induced a G2 + M phase arrest in 48-h exposed confluent BPAEC that persisted for at least 28 d and was associated with continued cellular enlargement. A short-duration MCTP exposure of confluent (low and high concentration) and log phase (high concentration) BPAEC caused persistent cell-cycle arrest for 1 wk, whereas a low-concentration exposure in log phase cells resulted in cell-cycle arrest with reversal 96 h after exposure. Western blot examination revealed that by 24 h of MCTP exposure, the phosphorylation state of cdc2 was consistent with the inactive form of the kinase (confirmed by biochemical assay); this alteration persisted through at least 96 h of exposure. We conclude that MCTP induces a progressive irreversible endothelial cell dysfunction leading to inactivation of cdc2 kinase and irreversible cell-cycle arrest at the G2 checkpoint.

Effect of monocrotaline metabolites on glutathione levels in human and bovine pulmonary artery endothelial cells.

After being dehydrogenated by cytochrome P450 enzymes in the liver, monocrotaline's highly-reactive pyrrole metabolite, dehydromonocrotaline, is believed to interact with pulmonary artery endothelial cells to initiate a pulmonary vascular toxicity resembling pulmonary hypertension. Glutathione, an abundant antioxidant in pulmonary artery endothelial cells, has been shown to react with and detoxify the pyrrolic metabolites derived from monocrotaline in the liver. Using high-performance liquid chromatography with electrochemical detection, glutathione levels were measured in a time- and dose-dependent manner in human pulmonary artery endothelial cells following treatment with dehydromonocrotaline, dehydroretronecine and N-ethylmaleimide and bovine pulmonary artery endothelial cells after treatment with dehydromonocrotaline. The bovine cells had 40% less glutathione than the human in the control groups. Bovine pulmonary artery endothelial glutathione levels were depleted 20% more than the human at 15 minutes when treated with 100 microM dehydromonocrotaline. 15 microM N-ethylmaleimide caused an 80% depletion of glutathione compared to a 30% depletion with 15 microM dehydromonocrotaline in human pulmonary artery endothelial cells.

Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14C-monocrotaline in rat liver microsomes.

The metabolism and covalent binding of 14C-monocrotaline in Sprague-Dawley (SD) rat liver microsomes was investigated using the inducers dexamethasone, clotrimazole, pregnenolone-16 alpha-carbonitrile, and phenobarbital. Monocrotaline is a pyrrolizidine alkaloid (PA) that causes a syndrome in rats that is a model for human primary pulmonary hypertension. It has been documented that bioactivation of PAs (dehydrogenation to reactive pyrroles) in the liver by cytochromes P450 is required for their toxicity. Covalent binding of these reactive pyrroles to tissue macromolecules has been hypothesized to correspond to PA toxicosis. We correlated metabolism and total microsomal covalent binding of 14C-monocrotaline with cytochrome P450 3A using the aforementioned inducers, troleandomycin (a cytochrome P450 3A inhibitor), erythromycin N-demethylase assay of cytochrome P450 3A activity, and Western blots employing anti-rat cytochrome P450 3A antibodies. In addition, autoradiography of membranes electroblotted from SDS-PAGE demonstrated the formation of radiolabeled adducts with specific protein(s). The most intensely radiolabeled protein bands have an apparent molecular weight of approximately 52 kDa, which was similar to the molecular weight detected by anti-rat cytochrome P450 3A antibodies in the Western blots. No radiolabeled proteins were detected in microsomes pretreated with troleandomycin.

Alteration in lung particle translocation, macrophage function, and microfilament arrangement in monocrotaline-treated rats.

Individuals with preexisting cardiopulmonary disease are thought to be more susceptible to acute episodes of particulate pollution resulting in increased morbidity and mortality. Our study was designed to evaluate particle fate and macrophage function in an animal model of monocrotaline (MCT)-induced pulmonary hypertension. Two weeks following a single MCT injection, Sprague-Dawley rats were exposed sequentially to two different colored fluorescent microspheres 1.0 micron in diameter by aerosolization. Morphometric evaluation of lung sections was performed 0 and 24 h following the final particle exposure to determine the intrapulmonary location of inhaled microspheres. A decrease in the number of particles phagocytized by alveolar macrophages and an increase of free particles overlying the epithelium were found in MCT-treated animals compared with control. Pulmonary macrophages recovered by bronchoalveolar lavage were evaluated for chemotactic and phagocytic ability. Macrophage chemotaxis was significantly impaired following MCT treatment compared with controls, whereas phagocytic activity of macrophages lavaged from MCT and control treatment groups was similar. Macrophages were stained for filamentous (F) and globular (G) actin using Texas-Red-labeled phalloidin and Oregon-green-labeled DNase I, respectively. The area of microfilament staining for F and G actin increased, but the ratio of F/G actin was significantly decreased in animals with MCT treatment compared with control. While the responses observed with MCT treatment, such as pulmonary edema, polymorphonuclear leukocytes influx, and unique macrophage morphology may contribute to impaired macrophage function, the change in microfilament arrangement suggests that MCT may inhibit macrophage chemotaxis and impair particle clearance from the lungs.

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.

Monocrotaline metabolism and distribution in Fisher 344 and Sprague-Dawley rats.

The metabolism and distribution of 14C-monocrotaline in Fisher 344 (F344) rats was compared with that in Sprague-Dawley (SD) rats. In vitro microsomal preparations, in situ isolated perfused livers and in vivo excretion and distribution studies were used to discern any differences between these two strains. These strains have previously been shown to differ in their susceptibility to monocrotaline-induced pulmonary hypertension. Hepatic phase I metabolism appears to be similar in both strains with N-oxidation and dehydrogenation to the reactive pyrroles as the major pathways. During the liver perfusions, SD rats generated more monocrotalic acid than F344 rats, but the microsome and excretion studies demonstrated no significant differences in the amount of monocrotalic acid. Monocrotalic acid is a stable byproducer of dehydromonocrotaline reacting with cellular nucleophiles and indicates the amount of monocrotaline dehydrogenation when carboxylesterase activity is negligible. These data suggest that the differences in strain susceptibility to pulmonary vascular toxicity is most likely due to differences in their response to the toxic metabolites.

Comparative cytotoxicity of monocrotaline and its metabolites in cultured pulmonary artery endothelial cells.

Metabolites of the pyrrolizidine alkaloid monocrotaline cause progressive development of pulmonary hypertension in rats. The putative reactive intermediate monocrotaline pyrrole (MCTP) has been shown to cause cytotoxicity, hypertrophy, decreased proliferation, and altered synthetic capability in cultured pulmonary endothelial cells. We compared effects of monocrotaline (MCT) at 60 micrograms/ml (0.185 mM) with previously identified metabolites, MCTP 10 micrograms/ml (0.031 mM) and glutathione-conjugated dihydropyrrolizine (GSH-DHP) 60 micrograms/ml (0.135 mM), in cultured bovine pulmonary artery endothelial cells (BPAECs). To determine whether endothelial metabolism might contribute to the mechanism of this toxicity, we used markers of cytotoxicity (LDH release), synthetic activity (PGI2 synthesis), hypertrophy (planimetry), cell density (cell count/area), and Evans blue albumin (EBA) transudation as a marker for loss of fluid barrier integrity. We found changes in all endothelial markers with MCTP only. MCTP caused increased LDH release by 48 hr, augmented PGI2 synthesis by 96 hr, and resulted in hypertrophy and decreased cell density by 48 hr that persisted at least 21 days. There was increased EBA transudation at 24 hr posttreatment. We concluded that, based on markers of endothelial damage, BPAECs showed no apparent ability to metabolize MCT to a reactive intermediate nor to further metabolize GSH-DHP to a toxic species. We also concluded that MCTP can cause a direct effect on fluid barrier integrity of endothelial cell monolayers in the absence of inflammation.

Cell cycle alterations associated with covalent binding of monocrotaline pyrrole to pulmonary artery endothelial cell DNA.

In the monocrotaline (MCT) rat model of pulmonary hypertension, the pulmonary vascular endothelium is thought to be the early target of the bifunctionally reactive metabolite monocrotaline pyrrole (MCTP). In previous studies, bovine pulmonary arterial endothelial cells (BPAEC) exposed to MCTP exhibited inhibition of proliferation. Since other compounds that crosslink DNA lead to cell cycle alterations, we utilized BPAEC to correlate the effects of MCTP on the cell cycle with the extent of covalent binding of [14C]MCTP to BPAEC DNA. Dose response (0.0 to 50.0 micrograms MCTP/ ml) and 96-hr time course (5 micrograms MCTP/ml low dose or 34.5 micrograms MCTP/ml high dose) studies were carried out followed by flow cytometric cell cycle analysis. High concentrations of MCTP caused cell cycle arrest in S phase, beginning by 24 hr, while an S phase delay was observed at low concentrations, but progressed to a G2 + M phase arrest by 48 hr. Covalent DNA binding (34.5 micrograms/ml of [14C]MCTP incubated with BPAEC) occurred within 1 hr and progressively increased through 96 hr. In conclusion, covalent binding of MCTP to DNA is associated with cell cycle arrest; however, the position of cell cycle arrest is dependent on dose, with an S phase arrest at high concentrations and a G2 + M phase arrest at low concentrations of MCTP. The mechanism by which MCTP induces proliferative inhibition could be cell cycle arrest.