Critical Reviews in Toxicology, 22(5/6):307-325 ( 1992)

Mechanisms and Pathology of Monocrotaline Pulmonary Toxicity D. W. Wilson,’* H. J. Segall121. C. Pan12M. W. Lame12J. E. and D. Morin2 Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of Toronto on 11/18/14 For personal use only.

Departments of Pathology’ and Pharmacology and Toxicology,* College of Veterinary Medicine, University of California-Davis, Davis, CA, 95616 *

To whom all correspondence should be addressed

ABSTRACT: Monocrotaline (MCT) is an 1 1-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 hepatoxic 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 I1 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.

KEY WORDS: monocrotaline, pulmonary hypertension, pyrrolizidine alkaloids, pulmonary pathology.

1. INTRODUCTION 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. Although MCT pneumotoxicity is widely used as a model to study the pathogenesis of human pulmonary hypertension, the toxicological mechanisms by which MCT initiates lung toxicity are unclear. Based on recent work in our and other laboratories, we have formulated the

following general hypotheses of the initiating mechanism in MCT pneumotoxicity . 1

2

3

Monocrotaline is activated to a reactive metabolite(s) in the liver. The selectivity of MCT for the lung, as opposed to that of other primarily hepatotoxic PAS, is a consequence of the differences in hepatic metabolism and blood kinetics of MCT. A likely candidate for a reactive metabolite is the dehydrogenation product monocrotaline pyrrole (MCTP).

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4. 5.

6.

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7.

Secondary or phase I1 metabolism of MCT is through glutathione (GSH) conjugation, which represents a detoxification reaction. A reactive hepatic metabolite(s) is accumulated by red blood cells (RBCs) where it is stabilized during transport to the lung. The pulmonary injury or early response is dependent on an inflammatory response that includes a significant role for platelet activation. Reactive hepatic metabolites cause noncytotoxic, but irreversible, endothelial injury.

The purpose of this review is to present the MCT model of pulmonary hypertension in relation to the human syndrome, to summarize the published work from which the above hypotheses have beeri derived, and iu present the as-yetunanswered questions that then arise.

II. OVERVIEW OF HUMAN PULMONARY HYPERTENSION AND THE MOST FREQUENTLY USED ANIMAL MODELS Pulmonary hypertension as a clinical syndrome of humans occurs as an “acute” or “chronic” disease. Abrupt elevation of pulmonary arterial pressure is usually, but not necessarily, caused by pulmonary embolism, which is often accompanied by acute right ventricular failure. Chronic pulmonary hypertension is a progressive disease of the pulmonary vascular wall with a multitude of causes and uncertain pathogenesis. Exposure to MCT is a documented cause of chronic pulmonary hypertension in animals’.2 and is frequently used as an experimental model to study the pathogenesis of this syndrome. Other clinically distinct syndromes of pulmonary hypertension are described in the following subsections.

A. Primary Pulmonary Hypertension Primary pulmonary hypertension is an enigmatic disorder found predominantly in young women, but it also affects a significant number of middle-aged and elderly males and females. It is characterized by elevated pulmonary arterial

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pressure in the absence of primary cardiac, parenchymal pulmonary, or systemic disease. It affects small pulmonary arteries, in which proliferative lesions involving endothelial cells, smooth muscle cells, and fibroblasts obstruct flow. Currently, primary pulmonary hypertension is classified into three subtypes based on clinical findings and presumed pathophysiology: plexogenic pulmonary arteriopathy (PPA), thromboembolic pulmonary hypertension (TPH), and veno-occlusive disease. The most common pathological findings include medial hypertrophy, thrombosis, intimal fibrosis, and plexiform l e s i o n ~While .~ dramatic progress in the characterization and diagnosis of this disease has been made since the first description of pulmonary arteriosclerosis of unknown origin over a century ago,4 treatment of this disease remains iargeiy paiiiative and affected patients progress to death from heart failure unless a hewlung transplant can be performed. Patients in the early stages of the disease are treated with calcium channel blocker vasodilatory agents and anticoagulant therapy. More critically ill patients can be treated with PGI, infusions and NO (endothelium-derived relaxing factor or EDRF) inhalation5 until transplantation can be performed. Recent success in lung transplant therapy has been reported but the expense and risk of rejection remain.6 The lack of curative therapy reflects the lack of a mechanistic knowledge of the initiating events in this disease. The hypothesis that dysfunction of endothelial cells contributes to the pathogenesis of primary pulmonary hypertension has been a recent focus of research. It has been shown, for instance, that PPA is associated with abnormalities of endothelial structure and function that could result in impaired release of EDRF, while TPH is brought about by cell injury that facilitates coagulation and thrombosis formation in the pulmonary vasculature. Growing evidence in recent years indicates that the balance of a variety of vasoregulatory mediators, many derived from endothelial cells, plays an important role in determining the vascular tone and disease progress i o n . ’ ~EDRF ~ is a very potent vasodilator and inhibitor of platelet function. It has been shown to be released from a variety of human blood vessels and to dilate human pulmonary arteries in ~ i t r oProstacyclin, .~ another endothelium-

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derived product, can evoke vasodilation and inhibition of platelet aggregation. l o Endothelin, a peptide synthesized and released from the endothelial cells, has profound vasoconstrictor properties in arteries and particularly in veins.” There may be a number of additional endothelium-derived contracting factors produced in human vasculature. Endothelial cells also synthesize and secrete a variety of growth factors that control the cellular dynamics of vascular smooth muscle cells.’2.’3More complete reviews of primary pulmonary hypertension are available. I 4 . l 5

B. Secondary Pulmonary Hyptersion Elevated arterial pressure in the pulmonary circulation appears to be a common outcome of a majority of chronic lung diseases, including, but not limited to, chronic bronchitis, emphysema, bronchiectasis, extensive tuberculosis, pneumoconiosis, cystic fibrosis, idiopathic fibrosis, and sarcoidosis. Other disorders associated with pulmonary hypertension include cardiovascular disease (e.g., mitral stenosis and many congenital heart diseases), neuromuscular disease (e.g., malfunctioning chest bellows), and central nervous disease (e.g., inadequate ventilatory drive from the respiratory center - “Ondine’s curse”). The most important cause of pulmonary hypertension, both clinically and economically, is chronic obstructive pulmonary disease (COPD). According to estimates by Witschi and Last,I6 over $12 billion direct and indirect loss per year results from COPD. Regardless of the etiology, arterial hypoxemia and respiratory acidosis appear to be major contributors to vasoconstriction and vascular remodeling, eventually leading to pulmonary hypertension and right ventricular hypertrophy (Refer to Klinger and Hill,” Palevsky and Fishman,I8 and Murphy et a1.I9 for complete reviews).

C. Animal Models of Pulmonary Hypertension The pathogenesis of pulmonary hypertension is a matter of considerable scientific interest and debate. The critical anatomic manifestations of

the disease occur in the medium to small (200 to 50 pm) pulmonary arterioles and include extension of medial smooth muscle into the smaller arterioles20 and alterations in the synthesis of extracellular matrix in the arteriolar media.2’ Animal models of primary pulmonary hypertension can be categorized into those induced by hypoxia, consequences of agents resulting in ARDS-like syndromes and the MCT rat model. Hypoxia appears to be the most “pure” model of primary vascular disease as it does not require systemic administration of a toxin or bioactive agent. While the vascular response to hypoxia is proposed to represent a physiological reaction to low alveolar oxygen tension, the receptors and intermediates directing the vascular response have not been definitively characterized. The hypoxia model has the disadvantage of difficult and expensive animal manipulations over a time course that precludes identification of an initiating time point. A significant component of ARDS includes elevations of pulmonary artery pressure.22Models of ARDS are generated by systemic administration of agents leading to diffuse alveolar damage, either type I cell toxins such as alphathion a p h t h ~ u r e aand ~ ~ 3-methylind0le,~~ or agents promoting intracapillary inflammation such as e n d ~ t o x i nor ~ ~platelet-activating .~~ factor.*’ The difficulty with these models is that the inflammatory and reparative response of the alveolar capillaries is difficult to separate from responses causing vascular lesions. MCT pneumotoxicity has several practical advantages as a model of primary hypertension. A single subcutaneous administration of an apparently rapidly eliminated compound leads to progressive pulmonary vascular hypertension with striking vascular lesions but without apparent cytotoxicity or destruction of alveolar capillaries. Lesions resulting from MCT treatment include medial hypertrophy and extracellular matrix secretion in pulmonary arteriesZ6 and increased amounts of smooth muscle in pulmonary arteriole^^^^^^ similar to that in primary pulmonary hypertension. This involvement of small (50to 70-pm diameter) arterioles is important in that this is the crucial restrictive site in PPA. MCTtreated rats do not, in the usual experimental design, demonstrate the plexiform dilatations of small arteries characteristic of the late stage of

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the human disease. This may reflect the relatively short time course (3 weeks) in most murine experiments currently described. The potential involvement of platelets and platelet thrombi (described below) suggests similarities in the mechanisms of MCT-induced disease and TPA. Pulmonary phlebitis and muscular hypertrophy similar to that described in human pulmonary veno-occlusive syndromes also occur in MCTtreated In primary pulmonary hypertension, the relative contribution of increased vascular smooth muscle and contractility vs. increased matrix and loss of vessel wall compliance to the elevated pulmonary artery pressure is uncertain. The regulation of smooth muscle proliferation and altered phenotypic expression is also uncertain. Roles for mediators derived from circulating inflammatory cell^^'.^^ and endothelial cells33to direct smooth muscle cell changes have been suggested. The MCT model has provided additional clues as to the pathogenesis of medial changes in pulmonary hypertension. Early descriptions of MCT-induced pulmonary pathology recognized a significant perivascular mononuclear inflamm a t i ~ nWe . ~ have ~ further characterized this process as an accumulation of macrophages in the adventitia in MCT-treated rats. This raises an additional alternative of smooth muscle cell modulation resulting from inflammatory mediators derived from the advential sheath rather than those derived from endothelial cells.29One of the earliest changes after MCT treatment is the increase in extracellular space in the adventitia of MCTtreated rats, which likely represents accumulation of edema fluid from altered microvascular endothelial cell ~ e r m e a b i l i t y . ~In ~ vitro e ~ p e r i r n e n t sdemonstrate ~~ marked cytologic alterations in MCTP-treated pulmonary endothelial cells, which might account for this increased permeability. These findings suggest a hypothesis that there is a role for altered lung fluid flux resulting from increased arteriolar or microvascular permeability in the initiation of the vascular response and that this mechanism may be common to pulmonary hypertension induced by a variety of inflammatory and chemically induced lung diseases.

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111. CLINICAL SYNDROMES ASSOCIATED WITH PYRROLlZlDlNE ALKALOID TOXICITY The PAS consist of a large group of natural plant products widely distributed botanically and geographically around the world. Over 200 PAS have been identified, coming from at least 300 plant species, and more than 13 fa mi lie^.^.^' Among over 60 genera, Crotalaria, Symphytum, Heliotropium, and Senecio are of most importance both economically and toxicologically. Many of these plants are used as sources of food, coffee substitutes, herbal teas, and medicine. Human poisoning has been documented due to eating the plants or plant products. PAS have been found in meat, honey, and milk but at levels uniikeiy to result in human toxicity. L.’x Structurally, PAS are derivatives of hydroxylated 1methyl-pyrrolizidine, a structure called the necine base, which contains two fused five-membered rings with a nitrogen at the fourth position. They can be categorized as nonester (alcohol), monoester, diester, and macrocyclic diester, exemplified by retronecine, heliotrine, lasiocarpine, and MCT, respectively (Figure 1). Only those PAS that contain a 1,2 double bond are known to be toxic to mammals. Human intoxication from PAS has been a serious problem in third world c o u n t r i e ~ . ~PA~-~’ as weeds in food crops containing plants can grow such as wheat or corn and are harvested with the grain. Several documented human epizootics of chronic liver disease have been associated with PA ingestion, including wheat contaminated with Heliotropium in A f g h a n i ~ t a n ,‘‘bush ~~ tea” ingestion in Jamaica,42 and Senecio-contaminated bread in South PAS have been shown to be present in honey in the U.S. and A ~ s t r a l i a . PAS ~ . ~ ~derived from S. longilobus (thread leaf groundsel) have resulted in toxicities associated with herbal teas in the U.S.46-48Hepatic veno-occlusive disease due to Crotalaria ingestion was shown to be responsible for 67 cases with 28 deaths among 486 villagers in India.40 The most frequent outcome of PA toxicity, in either humans or animals, is hepatic injury.

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age only when hepatoprotective manipulations prevent death from liver Not all species are equally susceptible to pulmonary toxicity induced by MCT. Crotalaria poisoning in pigs and poultry results largely in renal t o ~ i c i t y . ~ ~ . ~ * MCT-treated monkeys develop hepatic veno-occlusive d i e s a ~ eas , ~well ~ as pulmonary vascular disease.5oWith one possible exception, all clinical intoxications of humans caused by ingestion of MCT-containing plants have resulted in primary chronic liver disease.2 Human PA intoxications also differ from experimental and livestock poisonings in that human cases do not have the megalocytic hepatocytes that are characteristic of PA exposure in other species.2

0 I CH2

IV. HISTORICAL USE OF MCT TO INDUCE PH IN EXPERIMENTAL PATHOLOGY Lasiocarpine

Monocrotaline

FIGURE 1. Structure of PAS representative of the nonester (alcohol), monoester, diester, and macrocyclic diester classes as exemplified, respectively, by retronecine, heliotrine, lasiocarpine, and monocrotaline.

Acute toxicity from high doses of PAS results in periacinar hepatic necrosis. Most commonly, clinical intoxications result from chronic exposure and are characterized by random individual hepatocyte necrosis, hepatocellular megalocytosis, hepatic fibrosis, and veno-occlusive disease leading to portal h y p e r t e n ~ i o n . MCT ~ ~ . ~ ~is of interest because, in rats, it causes acute periacinar hepatic necrosis only at high doses while a single, lower-dose, treatment reproducibly causes pulmonary hypertension (PH) and cor pulmonale, but not hepatic pathology. Similar pulmonary vascular disease has been reproduced experimentally in monkeys,so and acute pulmonary edema has been shown to occur in MCT-treated dogs.5’ This apparently selective pulmonary toxicity is fairly unique to MCT and f u l ~ i n ewhile ,~~ other PA intoxications are manifest primarily as chronic liver d i s e a ~ e . ~ ~Other * ’ ~ -PAS, ~ ~ such as retrorsine, have been shown to cause lung dam-

MCT has been used as an experimental model of PH for over 25 years since the inducement of cor pulmonale in rats by Turner and Lalich.m A variety of dosing strategies and pathologic evaluations resulted in quite similar descriptions of pulmonary pathology resulting from MCT treatment. 34.60-64 The pathological changes are mild to moderate in the alveolar parenchyma and marked in the pulmonary vasculature. In the parenchyma, early changes include edema of the alveolar interstitium and, in high doses, alveolar edema and hemorrhage, which imply an early increase in capillary permeability. The alveolar interstitium at later times post-treatment becomes moderately thickened by increased extracellular matrix. A characteristic change in MCT toxicity is megalocytosis of type I1 alveolar epithelial cells.35This is apparent as early as 1 week posttreatment. Increased numbers of similarly enlarged alveolar macrophages also are seen.65 Vascular changes have been described both in large, bronchus-associated branches of the pulmonary artery and in arterioles in the alveolar parenchyma. Larger pulmonary arteries have adventitial edema and mononuclear inflammatory cell accumulations and media thickened by increased amounts of extracellular matrix with re-

31 1

in MCTP-treated bovine pulmonary endothelial duplication of elastic lamellae. Adventitial edema cells.36 and inflammatory cell accumulation also are A variety of experimental manipulations alter present in arterioles, but the medial changes genthe progression of MCT-induced vascular lesions erally are described as an “extension” of smooth to pulmonary hypertension. Diet restriction, parmuscle into previously nonmuscularized arterticularly the restriction of protein, lessens the ioles. 30 Plexiform lesions seen in human primary Juvenile rats are more hypertensive response. hypertension generally are not described. Intraresistant than adult rats to the effect of MCT.99 vascular thrombi are reported inconsistently. Coadministration of the ornithine decarboxylase Numerous investigators have shown that a inhibitor difluoromethylornithine prevents the single injection of MCT, or its dehydrogenation hypertensive response.8o While the dietary and product MCTP, causes a short intense insult with age changes must be considered in experimental a rapid loss (via excretion or inactivation) of the design and may be related to polyamines and their starting compound.*68 The acute changes that control of cell proliferation, not enough is known result include injury to alveolar capillary endothelium and formation of pulmonary edema.65-69.70 about the action of MCT in the lung to ascertain whether these effects alter the initial chemical Relatively early ultrastructural investigation of interaction with lung cells or merely affect the the alveolar capillary endothelial response to MCT demonstrated a progressive increase in capillary progression of the vascular disease. permeability to high molecular weight tracers in the absence of endothelial necrosis.7oThe authors have found similar evidence of delayed permeV. EVIDENCE FOR CURRENT ability defect^,^' with little endothelial cell cyHYPOTHESESFORMECHANISMSOF totoxicity evident even at doses high enough to MCT PNEUMOTOXICITY elicit hepatic necrosis (unpublished data). PAS often are described as antimitotic agents. A. Liver is the Major Site of MCT This is proposed to occur through the crosslinking Activation to a Proximate Toxin capability of the bifunctional reactive sites on either side of the metabolically activated necine At the present time, the mechanism by which base. This crosslinking activity purportedly inMCT initiates lung toxicity is not well characterferes with the cell cycle and leads to the charterized. The current concept of MCT toxicity is acteristic cyto- and karyornegaly described as that MCT is metabolized in the liver to a reactive megalocytosis in the pathological evaluation of species (pyrrole?), which is transported to the A marked strucchronic PA hepatot~xicity.~’.’~ lung to initiate endothelial injury. Evidence for ture-activity relationship between the crosslinkthe role of liver metabolism includes (1) if MCT ing capability of a variety of PAS and their ability is perfused through an isolated lung preparation , to inhibit proliferation in v i m has been demonthen the suppression of 5hydroxytryptamine (5~ t r a t e d MCT . ~ ~ treatment induces characteristic HT) removal and metabolism (an activity of pulkaryomegalic type LI cells in vivo (Figure 2). 35*74 monary endothelial cell function) occur only when Despite an apparent strong proliferative stimulus MCT has previously been activated/metabolized suggested by a high thymidine labeling index 7 by perfusion through an isolated Iiver preparadays after treatment,75 minimal proliferation of tion;81 and (2) the demonstration that pretreattype I1 cells occurs and these cells also appear ment with the mixed function oxidase inhibitor to be under some form of mitotic i n h i b i t i ~ n . ~ ~ SKF-525A prevented development of pulmonary This suggests that direct PA interaction with pulhypertension after MCT treatment while phenomonary type I1 cells occurs in MCT pneumotoxbarbital induction of hepatic mixed-function oxicity. Whether this effect extends, in vivo, to idase enzymes augmented toxicity. 82-84 These other potentially more pathogenetically relevant findings are all indirect and do not exclude nonalkaloid-derived byproducts of hepatotoxicity inlung cells (such as endothelial cells) remains to be determined. In vitro experiments do, however, terfering with pulmonary function. No one has demonstrate a similar inhibition of proliferation shown that a hepatic metabolite derived from

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FIGURE 2. Pulmonary parenchyma from a Sprague Dawley rat treated 3 weeks previously with 60 mg/kg s.q. MCT. Cyto- and karyomegaly are evident in a type II epithelial cell (11). An interstitial cell is also karyomegalic (arrowhead). (Original magnification x 1110.)

MCT reaches the lung and interacts with lung tissue in vivo. Such experiments were not possible previously due to the lack of radiolabeled MCT and its derivatives. Using isolated organ systems and I4C-MCT, Pan et al. recently demonstrated that the covalent interaction of MCT with lung tissue occurs and is dependent on prior hepatic metabolism.8s

6. Selectivity of MCT for the Lung Is a Consequence of Differences in Hepatic Metabolism and Blood Kinetics MCT is fairly selective in producing pulmonary insult at doses that for other PAS, such as senecionine (SEN), only produce hepatic lesions. The reactive pyrrolic metabolites of MCT are more stable than those of hepatoxic PAS,* potentially allowing greater amounts of hepaticgenerated reactive intermediates to reach the lung. However, this does not explain the lack of hepatic

toxicity at pneumotoxic doses of MCT. The difference in the structure of the acid moiety of MCT vs. SEN appears to facilitate marked changes in metabolism, tissue selectivity, and toxicity.86This selectivity also could be related to the differences in the distribution of these two PAS. The authors have compared the distribution kinetics of the PAS SEN and MCT to evaluate such differences. These kinetic studies have shown that both PAS are rapidly eliminated from the plasma, but are retained by RBCS.~~.*’ Whether the radioactivity retained in RBCs represents the parent compound or metabolites has not been determined. Experiments with isolated organ systems suggest that the material in RBCs is stabilized compared with that in plasma and is capable of covalent interaction with lung tissues (see later discussion). The retention of I4C-MCT equivalents in RBCs is substantially higher than that observed for I4CSEN (Figure 3). Other differences between the metabolism and kinetics of SEN and MCT also may alter the relative availability of these PAS to

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Concentration of MCT & SEN-equivalents in RBC's & Plasma Collection of bile/u rine/bl ood

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monocrotaline - RBC monocrotaline plasma senecionine - RBC senecionine - plasma

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50

0

0 t~ 0

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0

0 -25

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125 175 225 275 325 375 425 Time after dosing (minutes)

FIGURE 3. Comparison of blood kinetics of MCT and SEN after a single i.v. dose: there is a nearly parallel elimination curve for SEN and MCT in serum, but MCT has a markedly greater retention in the RBC fraction of blood.

lung tissue. These kinetic studies also suggest that only SEN is subject to enterohepatic recirculation. Major differences also exist in the types of metabolites formed. The authors found that senecionine N-oxide is a major metabolite in both the urine and bile of SEN-treated animals while, in the case of MCT, N-oxidation plays only a minor metabolic role. A large portion of administered MCT, in contrast to SEN, is excreted unchanged in the urine. Hayashi, using 'H-MCT, also found large concentrations of parent PA in the urine of rats.66The relative role of these later findings of divergent metabolism and elimination of these two PAS in selective organ toxicity remains to be determined.

C. Dehydromonocrotaline (Monocrotaline Pyrrole, MCTP) Is Most Likely the Reactive Intermediate The nature of the reactive MCT metabolite that circulates from the liver to the lung is un314

certain. Early work, based on hepatic microsomal metabolism, suggested the presence of a reactive MCTP has dehydrogenation product, MCTP. been considered to be a likely candidate for transport from the liver to the Colorimetric assays demonstrated pyrrolic compounds in the lung of MCT-treated rats.89 MCTP has been shown to suppress 5-HT removal from lung slicesgoand, when injected intravenously in the nonprotic solvent dimethylformamide (DMF), reproduces the pulmonary vascular syndrome induced by MCT. 19*67.91-101MCTP does not appear to require additional metabolism by mixed-function oxidases to affect the lung because pretreatment with either phenobarbital or SKF-525A did not affect the progression of MCTP-induced lung toxicity. ' 0 2 88989

D. MCTP Is Detoxified by Conjugation with Glutathione Recent research by our and other laboratories

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has demonstrated a significant role for GSH conjugation in the phase II metabolism of MCT by the liver.103.104 A summary of the proposed activation and phase II metabolic pathways for MCT is presented in Figure 4.The significance of conjugation of MCT with GSH and secondary metabolism in the genesis of lung toxicity remains to be determined. Using radiolabeled I4C-MCT in isolated perfused livers and whole animals, the authors recently identified two sulfhydryl-conjugated metabolites of 6,7-dihydro-7-hydroxy- 1hydromethyl-5H-pyrrolizine (DHP). From bile obtained in vivo from isolated perfused livers, the authors have isolated GSH-DHP and, from urine, an N-acetylcysteine conjugate (NAcysDHP) (Figure 4).Io3 To determine if thiol-conjugated pyrroles were important in the induction of pulmonary toxicity, the authors conducted parallel in vivo toxicity studies with GSH-DHP, cysteine-DHP (CYS-DHP), MCT, and MCTP. Doses were chosen based on the standard 60 mg/kg MCT used to induce pulmonary hypertension and the approximately 10- to 20-fold lower dose of

MCTP necessary to create similar lesions. Given the relatively low metabolic percentage seen in previous kinetic studies, it was estimated that a maximum 20% of the parent compound would potentially be available as glutathione conjugates. Rats were then dosed with the maximum estimated dosage (1 2 mg/kg) and twice the maximum dosage (24 mg/kg) of the primary GSHDHP conjugate. Animals given a single injection of MCT (60 mg/kg) developed pulmonary hypertension at the end of 3 weeks, as indicated by a significant elevation in right ventricular pressure. A parallel and significant increase in right ventricular weight ratios was also evident. Histopathology showed marked alterations both in pulmonary vasculature and parenchyma (Figure 5). Both high (5 mg/kg) and intermediate (3 mg/ kg) doses of MCTP led to severe pulmonary inflammation and hemorrhage, resulting in death within 2 weeks of treatment. A lower dosage of MCTP (1 mg/kg) caused a significantly elevated right ventricular pressure and an increased right ventricular weight ratio. Neither high (24 mg/kg)

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FIGURE 4. Proposed pathways for the metabolism of 14C-MCT (A) as determined from isolated perfused rat Hydrolysis by hepatic carboxylesterases does not appear to be a major pathway in the rat due to the inability to find the metabolite retronecine (C). MCT N-oxide (D) represented 4% of the recoverable I4C from perfusate in isolated perfused liver experiments. Trace amounts of DHP (F) were detected. Dehydrogenation to MCT pyrrole (E) is considered to be a major pathway. MCT pyrrole has been found to react with nucleophiles such as GSH to generate monocrotalic acid (B) and GSH-conjugated DHP. Identification of N-acetylcysteine-conjugatedDHP in the urine of rats dosed with MCT76indicates that GSH-DHP can be additionally acted upon by the enzymes gammaglutarnyltranspeptidase (1), cysteinylglycinase (2),and N-acetyltransferase (3). (Modified and reprinted from Reference 104.)

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A

B FIGURE 5. Pulmonary arterioles and parenchyma from rats treated 3 weeks previously with either saline (A), 24 mg/kg i.v. GSH conjugate of DHP (B), 1 mg/kg i.v. MCTP (C), or 60 mg/kg s.q. MCT (D). Arterioles from both MCT- and MCTP-treated rats have prominent muscular hypertrophy of the media and thickened sclerotic adventitia resulting in diminished lumen diameter. These animals also have slightly thickened alveolar septae and enlarged type II epithelial cells compared with either saline-treated controls or animals given GSH-DHP. (Original magnification x 488.)

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FIGURE 5D

nor low (1 2 mg/kg) dosages of DHP-GSH caused a detectable change in RVP or RV/LV + S, and no significant structural alteration in the lung was observed in these two treatment groups. The CYSDHP conjugate also failed to result in lung injury at a dose of 12 rng/kg. These studies suggest that free MCTP is pneumotoxic at the dosages tested while the GSH conjugates appear to represent detoxification products.

E. A Reactive Hepatic Metabolite@)Is Accumulated in RBCs Where It Is Stabilized during Transport to the Lung The high reactivity of MCTP, its rapid breakdown in aqueous Solutions,''' and the need for either direct injection in isolated l u n g preparation^^^ or use of DMF for it to affect the lung in viv0'05 raise the question of how the re317

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active MCT metabolite(s) are transported from the hepatocyte to the lung in vivo. It is also uncertain how much MCTP synthesized in the liver is accessible to lung tissue. Colorimetric assays suggested that MCTP in vitro has an approximately 5-s half-life in serum.Io2There is a possibility that, under in vivo conditions, the halflife of MCTP, or some other electrophilic metabolite, might be extended through an association with R B C S . The ~ ~ pharmacokinetic and distribution studies using 14C-MCT of Estep et al. demonstrate significant sequestration of radioactivity in RBCs 24 h after MCT injection, well after radioactivity in the plasma has declined. Io6 Further evidence that this RBC sequestration may represent an important transport phenomenon comes from experiments demonstrating RBCs containing MCT and/or metaboiites stabilize, for up to 2 h, an activity capable of suppressing 5HT and augment covalent binding of MCT metabolites in isolated perfused lung preparations (Figure 6).85The nature of the radiolabeled material sequestered in the RBC is unknown. Given that less, but still significant, covalent binding to lung occurred even in nonRBC-supplemented tandem lung preparations, it

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FIGURE 6. Covalent binding of I4Cderived from MCT to lung in isolated perfused liver and lung preparations (n = 5 per group). Livers were perfused with 400 pA4 14C-labeledMCT with and without RBCs followed by perfusion of isolated lungs by perfusate (Buffer-Liver) or RBCs reisolated from liver perfusate (RBC-Liver). Tandem and tandem-RBC experiments are similar preparations but with simultaneous perfusion of both liver and lung in series (see Reference 85 for details). (Reprinted from Pan, L. C., Lame, M. W., Morin, D., Wilson, D. W., and Segall, H. J., Toxicol. Appl. Pharmacol., 1 10, 336, 1991. With permission.)

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also remains uncertain what relative contribution the as-yet-uncharacterized material in RBCs makes to the amount of reactive metabolites available to interact with the lung in vivo.Whether this potential mechanism of transport represents a passive or active process of stabilization of reactive intermediates also is unknown. Alternatives for active processes of transport include secondary metabolism and transport of GSH conjugates out of the RBC (as has been demonstrated for l-chloro-2,4-dinitrobenzene[CDNB]Io8-’I ) with a possible secondary metabolism in the lung.

F. The Pulmonary Injury or Early Response Is Dependent on an Inflammatory Response That Includes a Significant Role for Piatelet Activation The nature of the interaction of MCT or its metabolites with the lung remains unclear. Several lines of evidence point to a role for inflammation in MCT toxicity. Anatomic evidence of inflammatory responses include the aforementioned mononuclear v a s ~ u l i t i s ~ and ~ . ~the ~ description of intravascular platelet thrombi in some studies of the post-MCT response. Other evidence is largely indirect. A variety of stressors, including dietary restriction, ’ I 2 steroid treatment, I I 3 and placebo injections,’ l 4 diminish the hypertensive response to MCT treatment. The macrophage-derived inflammatory mediator interleukin- 1 is increased in the bronchiolo-alveolar lavage fluid of MCT-treated rats.”’ Attempts to alter the immune response did not alter MCTPinduced toxicity, suggesting that the inflammatory reaction is not driven by hypersen~itivity.~~ Antioxidant treatment also failed to prevent MCTP-induced hypertension, implying that reactive oxygen species derived from inflammatory cells are not important in initial injury or progression.116The role of platelets in MCT pneumotoxicity has been investigated extensively. MCT treatment causes thromb~cytopenia,~~ and MCTP treatment results in accumulation of platelets in the lung.98 Platelet depletion appears to prevent the progression of MCTP-induced vascular disease.94The mechanism by which platelets affect the progression of vascular disease has not been definitively characterized. Levels of 5-HT and thromboxane, both vasoactive inflam-

endothelial cell necrosis plays a role in eliciting MCT-induced vascular disease remain uncertain. It is also unknown whether MCT acts locally to alter pulmonary artery endothelial cells and consequently stimulates local effects on medial smooth muscle. Alternatively, MCT may act at the alveolar capillary endothelium to interfere with alveolar capillary permeability. The vascular responses would then be a consequence of either G. Reactive Hepatic Metabolites Cause increased interstitial and perivascular fluid flux l 8 Noncytotoxic, but Irreversible, or resistance changes due to interstitial fibrosis Endothelial Injury and/or restriction of septal blood flow. Many aspects of MCT pneumotoxicity and its relationship to general mechanisms of pulBecause endothelial cells of the pulmonary hypertension remain to be determined. microvasculature have active b i o c h e m i ~ a l , ~ . ' ~ .monary ~~ Significant remaining questions include immunological, and surface-receptor"* activities modulating inflammatory responses, it seems logical that the inflammatory component of MCT Why the delay in onset of anatomically or toxicity may represent an alteration in endothelial physiologically detectable lung alterations cell function. Endothelial cells also are sources with a compound with a relatively short halfof potent mediators of smooth muscle cell milife of elimination? togenesis and phenotypic expression of matrix What is the role of GSH conjugation in proteins, both of which have been implicated in mechanisms of selective organ and cellular the pathogenesis of vascular disease due to toxicity with MCT? Does GSH conjugation MCT28*i19 and other models of pulmonary hyplay a similar role in the mechanism of PApertension. 120 Evidence that MCT treatment afinduced hepatotoxicity? fects endothelial cells includes (1) there is inDoes MCT primarily affect microvascular creased capillary permeability to large molecular or arterial endothelial cells? Do other lung weight tracers after MCT t ~ e a t m e n t ;(2) ~ ~there ,~~ cells directly affected by MCT also affect is increased 1251-albuminr e t e n t i o P and inthe progression of vascular disease? creased alveolar septal and vascular adventitial Does the demonstrated role of platelets reinterstitial space suggestive of edema fluid acflect initiation of or response to injury? Is cumulation in the early stages of MCT-induced the vascular response a consequence of mepulmonary toxicity (Figure 7);35(3) MCT treatdiator release from platelets or rather a conment depresses endothelial cell surface-associsequence of thrombosis and repair subseated enzyme activities such as angiotensin-conquent to local endothelial injury? verting e n ~ y m e , ' ~ ' -5-HT ' ~ ~ removal and What is the mechanistic connection bemetabolism,8' and plasminogen activator;'24 and tween the proposed endothelial cell injury (4)increased thymidine uptake by endothelial cells and the apparent arterial smooth muscle cell both in pulmonary arteries and veins occurs in response resulting in altered pulmonary cell turnover experiments in MCT-treated anihemodynamics? Is there a direct endothelial It remains to be determined mals (Figure 8).30*75 ce1l:smooth muscle cell interaction in arwhether MCT or its metabolites have a direct chemical interaction with endothelial cells or that terioles or is the vascular smooth muscle endothelial dysfunction is secondary to systemic response an indirect consequence of permeinflammation or the effects of MCT on other ability or hemodynamic alterations in the target lung cells. A recent report demonstrated a alveolar parenchyma? Is there a role for indirect effect of MCTP to cause karyomegaly in terstitial or perivascular fibrosis in eliciting cultured bovine pulmonary endothelial cells.36 The hemodynamic alterations stimulating the significance of this change in vivo and whether vascular smooth muscle response?

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matory mediators derived from platelet activation, did not change in MCTP-treated rats. Additionally, neither inhibitors of the receptors for 5-HT nor thromboxane antagonists alter the hypertensive response. l o l , iI 7

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FIGURE 7. Transmission electron micrograph of alveolar septa from a Sprague Dawley rat treated 96 h previously with 60 mg/kg s.q. MCT. There is separation of the endo- and epithelial cells at the blood-air barrier by extracellular edema fluid (arrowheads) in the absence of morphologically evident alterations in capillary endothelium. (Original magnification x 3100.) (Reprintedfrom Wilson, D. W. and Segall, H. J., Am. J. Patho/., 136, 1293, 1990. With permission.)

FIGURE 8. Autoradiograph of cell labeling from 3H-thymidineuptake experiments in a Sprague Dawley rat 4 days post-treatmentwith 60 mg/ kg MCT. An intraacinar arteriole is shown with silver grains overlying endothelial cells and a mononuclear cell in the arteriolar adventitia. (Original magnification x 1700.)

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In summary, the evidence that hepatic activation of MCT is required before lung injury can take place remains strong. There are several clues suggesting that metabolism and kinetics, as well as the relative stability of the reactive intermediates, direct selective organ toxicity among different PAS. While it seems possible the RBCs may play a role in stabilizing reactive intermediates, the mechanisms of transport between liver and lung remain incompletely characterized. MCT treatment appears to result in a microvascular permeability defect in the lung, but the nature of this interaction and the critical sites within the pulmonary vasculature are unknown. The pathophysiological connection between the purported endothelial dysfunction and the progression of vascular disease and the critical mediators regulating medial response have yet to be separated from the many proposed signals from endothelial and inflammatory cells, both in MCT pneumotoxicity and in human pulmonary hypertension.

REFERENCES 1. Cheeke, P. R., Toxicity and metabolism of pyrrolizidine alkaloids, J . Anim. Sci., 66, 2343, 1988. 2. Mattocks, A. R., Chemistry and Toxicology of Pyrrolizidine Alkaloids, Academic Press, New York, 1986. 3. Weir, E. K., The United States experience with the

acute and chronic treatment of primary pulmonary hypertension, Eur. Heart J . , (Sept. Suppl. J), 33, 1988. 4. Kanemoto, N. and Constant, J., Present status of

primary pulmonary hypertension, Tokai J . Exp. Clin. Med., 14(4), 257, 1989. 5. Dinh Xuan, A. T. D., Higenbottam, T. W., Scott, J. P., and Wallwork, J., Primary pulmonary hypertension: diagnosis, medical and surgical treatment, Respir. Med., 84, 189, 1990. 6. Trulock, E. P., Cooper, J. D., Kaiser, L. R., Pasque, M. K., Ettinger, N. A., and Dresler, C. M., The Washington University-Barnes Hospital experience with lung transplantation, JAMA, 266, 1943, 1991. 7. McCormack, D., Endothelium-derived relaxing fac-

tors and the human pulmonary circulation, Lung, 168 (Suppl.), 35, 1990. 8. Johns, R. A., Endothelium-derived relaxing factor: basic review and clinical implications, J . Cardiothorac. Vasc. Anesth., 5(1), 69, 1991.

9. Moncada, S., Palmer, R. M., and Higgs, E. A.,

Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev., 43, 109, 1991. 10. Dusting, G. J. and MacDonald, P. S., Prostacyclin and vascular function: implications for hypertension and atherosclerosis, Pharmucol. Ther., 48, 323, 1990. 11. Rubanyi, G . M. and Botelho, L. H., Endothelins, FASEE J . , 5(12), 2713, 1991. 12. Vender, R. L., Clemmons, D. R., Kwock, L., and Friedman, M., Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s), Am. Rev. Respir. Dis., 135(3), 622, 1987. 13. Hannan, R. L., Kourembanas, S., Flanders, K. C.,

Rogelj, S. J., Roberts, A. B., Faller, D. V., and Klagsbrun, M., Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta, Growth Factors, I ( ] ) , 7, 1988. 14. Rich, S. and Brundage, B. H., Pulmonary hypertension: a cellular basis for understanding the pathophysiology and treatment, J . Am. Coll. Cardiol., 14(3), 545, 1989. 15. Hawkins, J. W. and Dunn, M. I., Primary pulmonary hypertension in adults, Clin. Cardiol., 13(6). 382, 1990. 16. Witschi, H. and Last, J. A., Pulmonary toxicology, in Handbook of Toxicology, Haley, T. J . and Berndt, W. O., Eds., Hemisphere, Washington, D.C., 1987, 113. 17. Klinger, J. R. and Hill, N. S., Right ventricular

dysfunction in chronic obstructive pulmonary disease: evaluation and management, Chest, 99(3), 7 15, 1991. 18. Palevsky, H. I. and Fishman, A. P., Chronic cor

pulmonale: etiology and management, JAMA, 263( 17), 2347, 1990. 19. Murphy, M. L., Dinh, H., and Nicholson, D., Chronic cor pulmonale, Disease-A-Month, 35( lo), 653, 1989. 20. Wagenvoort, C. A. and Wagenvoort, N., Primary

pulmonary hypertension: a pathological study of the lung vessels in 156 clinically diagnosed cases, Circulation, 42, 1163, 1970. 21. Mecham, R. P., Whitehouse, L. A., Wrenn, D. S., Parks, W. C., Griffin, G. L., Senior, R. M., Crouch, E. C., Stenmark, K. R., and Voelkel, N. F., Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension, Science, 237, 423, 1987. 22. Snow, R. L., Davies, P., Pontoppidan, H., Zapol,

W. M., and Reid, L., Pulmonary vascular remodeling in adult respiratory distress syndrome, Am. Rev. Respir. Dis., 126, 887, 1982. 23. Michel, R. P., Smith, T. T., and Poulsen, R. S., Distribution of fluid in bronchovascular bundles with permeability lung edema induced by alpha-naphthylthiourea in dogs - a morphometric study, Lab. Invest., 51, 97, 1984.

321

Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of Toronto on 11/18/14 For personal use only.

24. Woods, L. W., Wilson, D. W., Schiedt, M. J., and Giri, S. N., Structural and biochemical changes in lungs of 3-methylindole-treated rats, Am. J . Parhol., in press. 25. Meyrick, B. and Brigham, K. L., Repeated Escherichia coli endotoxin-induced pulmonary inflammation causes chronic pulmonary hypertension in sheep, Lab. Invest., 5 5 , 164, 1986. 26. Meyrick, B. and Brigham, K. L., Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep - structure:function relationships, Lab. Invest., 48, 458, 1983. 27. Ohar, J. A., Waller, K. S., DeMello, D., and Lagunoff, D., Administration of chronic intravenous platelet-activating factor induces pulmonary arterial atrophy and hypertension in rabbits, Lab. Invest., 65, 451, 1991. 28. Todorovich-Hunter, L., Johnson, D. J., Ranger, P., Keeley, F. W., and Rabinovitch, M., Altered elastin and collagen synthesis associated with progressive pulmonary hypertension induced by monocrotaline: a biochemical and ultrastructural study, Lab. Invest., 58, 184, 1988. 29. Wilson, D. W., Segall, H. J., Pan, L. C. W., and Dunston, S. K., Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treatedrats, Microvasc. Res., 38, 57, 1989. 30. Meyrick, B. 0. and Reid, L. M., Crotalaria-induced pulmonary hypertension uptake of 3H-thymidine by the cells of the pulmonary circulation and alveolar walls, Am. J . Pathol., 106, 84, 1982. 31. Kelly, J., Cytokines of the lung, Am. Rev. Respir. Dis., 141, 765, 1990. 32. Gillespie, M. N., Rippetoe, P. E., Haven, C. A., Shiao, R.-T., Orlinska, U., Maley, B. E., and Olson, J. W., Polyamines and epidermal growth factor in monocrotaline-induced pulmonary hypertension, Am. Rev. Respir. Dis., 140, 1463, 1989. 33. Vender, R. L., Clemmons, D. R., Kwock, L., and Friedman, M., Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s), Am. Rev. Respir. Dis., 135(3), 622, 1987. 34. Lalich, J. J. and Merkow, L., Pulmonary arteritis produced in rats by feeding Crotalaria spectabilis, Lab. Invest., 10, 744, 1961. 35. Wilson, D. W. and Segall, H. J., Changes in type I1 cell populations in monocrotaline pneumotoxicity, Am. J . Pathol., 136, 1293, 1990. 36. Reindel, J. F., Chang, C. C., Madhukar, B. V., and Roth, R. A., Monocrotaline pyrrole induces antiproliferative and delayed cytotoxic responses in cultured cells, Lung Parhophysiol., Al181, 5142, 1988. 37. Smith, L. W. and Culvenor, C. C. J., Plant sources of hepatotoxic pyrrolizidine alkaloids, J . Nat. Products, 44, 129, 1981.

322

38. Bult, L. B., Culvenor, C. C. J., and Dick, A. T., The pyrrolizidine alkaloids, in Frontiers of Biology, North-Holland, Amsterdam, 1968. 39. Mohabbat, O., Younos, M. S., Merzad, A. A,, Srivastava, R.N.,Sediq, G. G., and Aram, G. N., An outbreak of hepatic veno-occlusive disease in northwestern Afghanistan, Lancet, 269, 1976. 40. Tandon, B. M., Tandon, R. K., Tandon, H. D., Narndranathan, M., and Joshi, Y. K., An epidemic of veno-occlusive disease of liver in central India, Lancet, 2, 271, 1976. 41. Schoental, R., Health hazards of pyrrolizidine alkaloids: ashortreview, Toxicol. Lett., 10,323, 1982. 42. Bras, G., Jelliffee, D. B., and Stuart, K. L., Venoocclusive disease of liver with non-portal type of cirrhosis occurring in Jamaica, Arch. Pathol., 57, 285, 1954. 43. McLean, E. K., The toxic actions of pyrrolizidine (Senecio)alkaloids, Pharmacol. Rev., 22,429, 1970. 44. Deinzer, M. L., Thomson, P. A., Burgett, D. M., and Isaacson, D. L., Pyrrolizidine alkaloids: their occurrence in honey from tansy ragwort (Senecio jacobaea L.), Science, 195, 497, 1977. 45. Culvenor, C. C. J., Edgar, J. A., and Smith, L. W., Pyrrolizidine alkaloids in honey from Echium plantagineum L, J . Agric. Food Chem., 29, 958, 1981. 46. Stillman, A. E., Huxtable, R., Consroe, P., Kohnen, P., and Smith, S., Hepatic veno-occlusive disease due to pyrrolizidine (senecio) poisoning in Arizona, Gastroenterology, 73, 349, 1977. 47. Huxtable, R., Stillman, A., and Ciaramitaro, D., Characterization of alkaloids involved in human senecio (pyrrolizidine) poisoning, Proc. West. Pharmacol. Soc.. 20, 455, 1977. 48. Huxtable, R. J., Herbal teas and toxins: novel aspects of pyrrolizidine poisoning in the United States, Perspect. Biol. Med., 24, I , 1980. 49. Shulman, H. M., Luk, K., Deeg, H. J., Shuman, W. B., and Storb, R., Induction of hepatic venoocclusive disease in dogs, Am. J . Pathol., 126, 114, 1987. 50. Allen, J. R. and Chesney, C. F., Effect of age on development of cor pulmonale in nonhuman primates following pyrrolizidine alkaloid intoxication, Exp. Mol. Pathol., 17, 220, 1972. 51. Miller, W. C., Rice, D. L., Kreusel, R. G., and Bedrossian, W. M., Monocrotaline model of noncardiogenic pulmonary edema in dogs, J . Appl. Physiol.9 45, 962, 1978. 52. Wagenvoort, W. C. A., Wagenvoort, N., and Dijk, H. J., Effect of fulvine on pulmonary arteries and veins of the rat, Thorax, 29, 522, 1974. 53. Ghodsi, F. and Will, J. A., Changes in pulmonary structure and function induced by monocrotaline intoxication, Am. J . Physiol.. 240, H149, 1981. 54. Barnes, J. M., Magee, P. N., and Schoental, R., Lesions in the lungs and livers of rats poisoned with

Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of Toronto on 11/18/14 For personal use only.

the pyrrolizidine alkaloid fulvine and its N-oxide, J. Pathol. Bacteriol., 88, 521, 1964. 5 5 . Molteni, A., Ward, W. F., Ts’ao, C., and Solliday, N. H., Monocrotaline-induced cardiopulmonary injury in rats; modification by the nonthiol ACE inhibitors CGS13945 and CGS16617, Arch. Int. Pharmacodyn., 291, 21, 1988. 56. Mattocks, A. R., Acute hepatotoxicity and pyrrolic metabolites in rats dosed with pyrrolizidine alkaloids, Chem.-Biol. Interact., 5, 227, 1972. 57. Hooper, P. T. and Scanlan, W. A., Crotalaria retusa poisoning of pigs and poultry, Aust. Vet. J . , 53, 109, 1977. 5 8 . Peckham, J. C., Sangster, L. T., and Jones, 0. H., Crotalaxia spectabilis poisoning in swine, JAVMA, 165, 633, 1974. 59. Allen, J. R., Carstens, L. A., and Olson, B. E., Veno-occlusive disease in Mucaca speciosa monkeys, Am. J . Pathol., 50, 653, 1967. 60.Turner, J. H. and Lalich, J. J., Experimental cor pulmonale in the rat, Arch. Pathol., 79, 409, 1965. 61. Merkow, L. and Kleinerman, J., An electron microscopic study of pulmonary vasculitis induced by monocrotaline, Lab. Invest., 15, 547, 1966. 62. Kay, J. M., Keane, P. M., and Suyama, K. L., Pulmonary hypertension induced in rats by monocrotaline and chronic hypoxia is reduced by p-chlorophenylalanine, Respiration, 47, 48, 1985. 63. Koletsky, A., Oyasu, R., and Reddy, J. K., Mutagenicity of the pyrrolizidine (Senecio) alkaloid lasiocarpine in the Salmonella/microsome test, Lab. Invest., 38, 352, 1978. 64, Kay, J. M. and Heath, D., Observations on the pulmonary arteries and heart weight of rats fed on Crotalaria spectabilis seeds, J . Pathol. Bacteriol., 92, 385, 1966. 65. Sugita, T., Hyers, T. M., Dauber, I. M., Wagner, W. W., McMurtry, I. F., and Reeves, J. T., Lung vessel leak precedes right ventricular hypertrophy in monocrotaline-treated rats, J . Appl. Physiol., 54, 371, 1983. 66, Hayashi, Y., Excretion and alteration of monocrotaline in rats after a subcutaneous injection, FASEB J., 25, 688, 1966. 67. Hilliker, K. S., Bell, T. G., and Roth, R. A., Pneumotoxicity and thrombocytopenia after single injection of monocrotaline, Am. J . Physiol., 242, H573, 1982. 68. Hsu, C. I., Allen, J. R., and Shumaker, R. C., Tissue distribution of tritium-labeled dehydroretronecine, Chon.-Biol. Interact., 8, 163, 1974. 69. Plestina, R. and Stoner, H. B., Pulmonary oedema in rats given monocrotaline pyrrole, J . Pathol., 106, 235, 1912. 70. Valdivia, E., Sonnad, J., Hayashi, Y.,and Lalich, J. J., Experimental interstitial pulmonary edema, Angiology, 18, 378, 1967.

71. Petry, T. W., Bowden, G. T., Huxtable, R. J., and Sipes, I. G., Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline, Cancer Res., 44, 1505, 1984. 72. Culvenor, C. C. J., Downing, D. T., Edgar, J. A., and Jago, M. V., Pyrrolizidine alkaloids as alkylating and antimitotic agents, Ann. N . Y . Acad. Sci., 163, 837, 1969. 73. Hincks, J. R., Kim, H.-Y., Segall, H. J., Molyneux, R. J., Stermitz, F. R., and Coulombe, R. A., DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure activity relationships, Toxicol. Appl. Pharmacol., 111, 90, 1991. 74. Mattocks, A. R. and White, I. N. H., Toxic effects and pyrrolic metabolites in the liver of young rats given the pyrrolizidine alkaloid retrorsine, Chem.Biol. Interact., 6, 297, 1973. 75. Wilson, D. W., Hacker, A. H., and Segall, H. J., Cell proliferation in the lungs of monocrotaline-treated rats, in Proc. 3rd Int. Symp. Poisonous Plants, James, L. F . , Keeler, R . F . , Bailey, E. M., Cheeke, P. R . , and Hegarty, M. P., Eds., Iowa State University Press, Ames, 1992, 225 76. Woods, L. W. and Wilson, D. W., Monocrotaline inhibition of 3-methylindole-induced type I1 cell hyperplasia, Proc. Am. Coll. Vet. Parhol., 30, 1989. 77. Ganey, P. E., Fink, G. D., and Roth, R. A., The effect of dietary restriction and altered sodium intake on the cardiopulmonary toxicity of monocrotaline pyrrole, Toxicol. Appl. Pharmacol., 78, 5 5 , 1985. 78. Hayashi, Y., Kato, M., and Otusuka, H., Inhibitory effects of diet-reduction on monocrotaline intoxication in rats, Toxicol. Lett., 3, 151, 1979. 79. Todd, L., Mullen, M., Olley, P. M., and Rabinovitch, M., Pulmonary toxicity of monocrotaline differs at critical periods of lung development, Pediatr. Res., 19, 731, 1985. 80. Olson, J. W., Atkinson, J. E., Hacker, A. D., Altiere, R. J., and Gillespie, M. N., Suppression of polyamine biosynthesis prevents monocrotalineinduced pulmonary edema and arterial medial thickening, Toxicol. Appl. Pharmacol., 81, 91, 1985. 81. Lafranconi, W. M. and Huxtable, R. J., Hepatic metabolism and pulmonary toxicity of monocrotaline using isolated perfused liver and lung, Biochem. Pharmacol., 33, 2479, 1984. 82. Mattocks, A. R., Toxicity and metabolism of Senecio alkaloids, in Phytochemical Ecology. Harborne, J. B . , Ed., Academic Press, New York, 1972, 179. 83. Allen, J. R., Chesney, C. F., and Frazee, W. J., Modifications of pyrrolizidine alkaloid intoxication resulting from altered hepatic microsomal enzymes, Toxicol. Appl. Pharmacol., 23, 470, 1972. 84. Mattocks, A. R., Oxidation of retronecine and synthanecine A to aldehydes, J . Chem. R e s . , S , 40, 1977. 85. Pan, L. C., Lame, M. W., Morin, D., Wilson, D. W., and Segall, H. J., Red blood cells augment

323

Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of Toronto on 11/18/14 For personal use only.

transport of reactive metabolites of monocrotaline from liver to lung in isolated and tandem liver and lung preparations, Toxicol. Appl. Pharmacol., 110, 336, 1991. 86. Estep, J. E., Lame, M. W., and Segall, H. J., Excretion and blood radioactivity levels following 14C-senecionine administration in the rat, Toxicology, 64, 179, 1990. 87. Estep, J. E., Lamb, M. W., Morin, D., Jones, A. D., Wilson, D. W., and Segall, H. J., 14Cmonocrotaline kinetics and metabolism in the rat, Drug Metab. Dispos., 19, 135, 1991. 88. Mattocks, A. R. and White, I. N. H., The conversion of pyrrolizidine alkaloids to N-oxides and to dihydropyrrolizine derivatives by rat-liver microsomes in vitro, Chem.-Biol. Interact., 3 , 383, 1971. 89. Mattocks, A. R., Toxicity of pyrrolizidine-alkaloids, Nature, 217, 723, 1968. 90. Hilliker, K. S., Garcia, C. M., and Roth, R. A., Effects of monocrotaline pyrrole on 5-hydroxytrypiiiliiinc iiiid pa-aquat uptAite by luiig slicite~, RCJ.Ccimmun. Chem. Pathol. Pharmacol., 40, 179, 1983. 91. Ganey, P. E., Sprugel, K. H., White, S. M., Wagner, J. G., and Roth, R. A., Pulmonary hypertension due to monocrotaline pyrrole is reduced by moderate thrombocytopenia, Am. J . Physiol., 255, H1165, 1988. 92. Ganey, P. E., Sprugel, K. H., Hadley, K. B., and Roth, R. A., Monocrotaline pyrrole-induced cardiopulmonary toxicity is not altered by metergoline or ketanserin, J . Pharmacol. Exp. Ther., 237,226, 1986. 93. Hilliker, K. S. and Roth, R. A., Increased vascular responsiveness in lungs of rats with pulmonary hypertension induced by monocrotaline pyrrole, Am. Rev. Respir. Dis., 131, 46, 1985. 94. Hilliker, K. S., Bell, T. G., Lorimer, D., and Roth, R. A., Effects of thrombocytopenia on monocrotaline pyrrole-induced pulmonary hypertension, Am. J . Physiol., 246, H747, 1984. 95. Bruner, L. H., Hilliker, K. S., and Roth, R. A., Cyclosporin A (CyA) and dexamethasone (DX) protect rats from the cardiopulmonary effects of monocrotaline pyrrole, Physiologist, 26, A-57, 1983. 96. Hilliker, K. S. and Roth, R. A., Injury to the isolated, perfused lung by exposure in vitro to monocrotaline pyrrole, Exp. Lung Res., 8, 201, 1984. 97. Bruner, L. H., Johnson, K. J., Till, G. O., and Roth, R. A., Complement is not involved in monocrotaline pyrrole-induced pulmonary injury, Am. J . Physiol., 254, H258, 1988. 98. White, S. M. and Roth, R. A., Pulmonary platelet sequestration is increased following monocrotaline pyrrole treatment of rats, Toxicol. Appl. Pharmacol., 96, 465, 1988. 99. Bruner, L. H., Johnson, K., Carpenter, L. J., and Roth, R. A., Lack of effects of deferoxamine, dimethyl sulfoxide, and catalase on monocrotaline pyrrole pulmonary injury, J . Toxicol. Environ. Health, 21, 205, 1987.

324

100. White, S. M., Wagner, J. G., and Roth, R. A.,

Effects of altered platelet number on pulmonary hypertension and platelet sequestration in monocrotaline pyrrole-treated rats, Toxicol. Appl. Pharmacol., 99, 302, 1989. 101. Ganey, P. E. and Roth, R. A., Thromboxane does not mediate pulmonary vascular response to monocrotaline pyrrole, Am. J . Physiol., 252, H743, 1987. 102. Bruner, L. H., Carpenter, L. J., Hamlow, P., and Roth, R. A., Effect of a mixed function oxidase inducer and inhibitor on monocrotaline pyrrole pneumotoxicity, Toxicol. Appl. Pharmacol., 85, 416, 1986. 103. Estep, J. E., Lame, M. W., Jones, A. D., and Segall, H. J., N-Acetylcysteine-conjugated pyrrole identified in rat urine following administration of two pyrrolizidine alkaloids, monocrotaline and senecionine, Toxicol. Lett., 54, 61, 1990. 104. Lame, M. W., Jones, A. D., Morin, D., and Segall, H. J., Metabolism of ['4C]monocrotaline by isolated ptxfti& rat i i v c i , Drug IVeiaL. D k p j . , i9, 516, 1991. 105. Bruner, L. H., Hilliker, K. S., and Roth, R. A., Pulmonary hypertension and ECG changes from monocrotaline pyrrole in the rat, Am. J . Physiol., 245, H300, 1983. 106. Estep, J. E., Lamb, M. W., Morin, D., Segall, H. J., and Wilson, D. W., 14C-Monocrotaline tissue distribution, covalent binding, and kinetics in the rat, in Proc. 3rd Int. Symp. on Poisonous Plants, James, L. F., Keeler, R. F., Bailey, E. M., Cheeke, P. R., and Hegarty, M. P., Eds., Iowa State University Press, Ames, 1992, 584. 107. Pan, L. C., Wilson, D. W., Segall, H. J., Lame, M. W., and Morin, D., Red blood cells from monocrotaline treated rats decrease serotonin removal in isolated perfused lungs, Toxicologist, 10, 101, 1990. 108. Awasthi, Y. C., Garg, H. S., Dao, D. D., Partridge, C. A., and Srivastava, S. K., Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on hemoglobin, Blood, 58, 733, 1981. 109. Awasthi, Y. C., Misra, G., Rassin, D. K., and Srivastava, S. K., Detoxification of xenobiotics by glutathione S-transferases in erythrocytes: the transport of the conjugate of glutathione and l-chloro-2,4dinitrobenzene, Br. J . Haematol., 5 5 , 419, 1983. 110. LaBelle, E. F., Singh, S. V., Srivastava, S. K., and Awasthi, Y. G., Dinitrophenyl glutathione efflux from human erythrocytes is primary active ATPdependent transport, Biochem. J . , 238, 443, 1986. 11 1. Awasthi, Y.C., Singh, S. V., Ahmad, H., Wronski, L. W., Srivastava, S. K., and LaBelle, E. F., ATP dependent primary active transport of xenobiotic-glutathione conjugates by human erythrocyte membrane, Mol. Cell. Biochem., 91, 131, 1989. 112. Ganey, P. E., Fink, G. D., and Roth, R. A., The effect of dietary restriction and altered sodium intake

Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of Toronto on 11/18/14 For personal use only.

on the cardiopulmonary toxicity of monocrotaline pyrrole, Toxicol. Appl. Pharmacol., 78, 55, 1985. 113. Hilliker, K. S. and Roth, R. A., Alteration of monocrotaline pyrrole-induced cardiopulmonary effects in rats by hydralazine, dexamethasone or sulphinpyrazone, Br. J . Pharmacol., 82, 375, 1984. 114. Langleben, D. and Reid, L. M., Effect of methylprednisolone on monocrotaline-induced pulmonary vascular disease and right ventricular hypertrophy, Lab. Invest., 52, 298, 1985. 115. Gillespie, M. N., Goldblum, S. E., Cohen, D. A., and McClain, C. J., Interleukin 1 bioreactivity in the lungs of rats with monocrotaline induced hypertension, Proc. SOC.Exp. Biol. Med., 187, 26, 1988. 116. Bruner, L. H., Bull, R. W., and Roth, R. A., The effect of immunosuppressants and adoptive transfer in monocrotaline pyrrole pneumotoxicity, Toxicol. Appl. Pharrnacol., 9 1, I , 1987. 117. Ganey, P. E., Hilliker-Sprugel, K., Hadley, K. B., and Roth, R. A., Monocrotaline pyrrole-induced cardiopulmonary toxicity is not altered by metergoline or ketanserin, J . Pharmacol. Exp. Ther., 237, 226, 1986. 118. Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A., Jr., and Seed, B., Endothelial leukocyte adhesion molecule. I. An inducible receptor for neutrophils related to complement regulatory proteins and lectins, Science, 243, 1160, 1989.

119. Meyrick, B., Gamble, W., and Reid, L., Development of crotalaria pulmonary hypertension: hemodynamic and structural study, Am. J . Physiol., 239, H692, 1980. 120. Stenmark, K., Crouch, E. C., and Parks, W. C., Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension and demonstrated by in situ hybridization, Am. J . Pathol., 135, 1073, 1989. 121. Hayashi, Y., Kokubo, T., Takahashi, M., Furukawa, F., Otsuka, H., and Hashimoto, K., Correlative morphological and biochemical studies monocrotaline-induced pulmonary alterations in rats, Toxicol. Lett., 21, 65, 1984. 122. Shale, D. J., Wiseman, M. S., and Cookson, W. 0.C. M., Effect of monocrotaline ingestion on the distribution of protein and angiotensin converting enzyme activity in the rat lung, Thorax, 41, 914, 1986. 123. Lafranconi, W. M. and Huxtable, R. J., Changes in angiotensin-converting enzyme activity in lungs damaged by the pyrrolizidine alkaloid monocrotaline, Thorax, 38, 307, 1983. 124. Molteni, A., Ward, W. E., Ts’ao, C., Port, C. D., and Solloday, N. H., Monocrotaline-induced pulmonary endothelial dysfunction in rats, Proc. SOC. Exp. Biol. Med.. 176, 88, 1984.

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