Perspective

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Pyrrolizidine Alkaloids: Potential Role in the Etiology of Cancers, Pulmonary Hypertension, Congenital Anomalies, and Liver Disease † ‡ § John A. Edgar,*, Russell J. Molyneux, and Steven M. Colegate † CSIRO Food and Nutrition, 11 Julius Avenue, North Ryde, NSW 2113, Australia ‡ Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, Hawaii 96720, United States § Poisonous Plant Research Laboratory, ARS/USDA, 1150 East 1400 North, Logan, Utah 84341, United States

ABSTRACT: Large outbreaks of acute food-related poisoning, characterized by hepatic sinusoidal obstruction syndrome, hemor- rhagic necrosis, and rapid liver failure, occur on a regular basis in some countries. They are caused by 1,2-dehydropyrrolizidine alkaloids contaminating locally grown grain. Similar acute poisoning can also result from deliberate or accidental consumption of 1,2- dehydropyrrolizidine alkaloid-containing herbal medicines, teas, and spices. In recent years, it has been confirmed that there is also significant, low-level dietary exposure to 1,2-dehydropyrrolizidine alkaloids in many countries due to consumption of common foods such as honey, milk, eggs, salads, and meat. The level of 1,2- dehydropyrrolizidine alkaloids in these foods is generally too low and too intermittent to cause acute toxicity. However, these alkaloids are genotoxic and can cause slowly developing chronic diseases such as pulmonary arterial hypertension, cancers, cirrhosis, and congenital anomalies, conditions unlikely to be easily linked with dietary exposure to 1,2-dehydropyrrolizidine alkaloids, especially if clinicians are unaware that such dietary exposure is occurring. This Perspective provides a comprehensive review of the acute and chronic toxicity of 1,2-dehydropyrrolizidine alkaloids and their potential to initiate certain chronic diseases, and suggests some associative considerations or indicators to assist in recognizing specific cases of diseases that may have resulted from dietary exposure to these hazardous natural substances. If it can be established that low-level dietary exposure to 1,2-dehydropyrrolizidine alkaloids is a significant cause of some of these costly and debilitating diseases, then this should lead to initiatives to reduce the level of these alkaloids in the food chain.

■ CONTENTS Notes 13 Abbreviations 13 1. Introduction 4 References 13 2. Toxicology 6 2.1. Occurrence, Evolution, and Chemistry 6 2.2. Bioactivation and Mechanism of Toxicity 6 1. INTRODUCTION 3. Pathological Effects 7 3.1. Sinusoidal Obstruction Syndrome and Cir- 1,2-Dehydropyrrolizidine ester alkaloids (dehydroPAs) have rhosis 7 been known for almost a century to be the cause of large and 3.2. Pulmonary Arterial Hypertension (PAH) 8 recurring episodes of acute hepatotoxicity in a number of − 3.3. Carcinogenesis 9 countries.1 16 The alkaloids responsible for these outbreaks of 3.4. Teratogenicity 9 poisoning are produced by weeds that often infest grain crops, 4. Possible Indicators of Dehydropyrrolizidine Alka- and the primary cause is consumption of bread made using loid Involvement 9 dehydroPA-contaminated flour. Typical infestations of dehy- 4.1. Cancer 9 droPA plants are shown in Figure 1. 4.2. Pulmonary Arterial Hypertension 10 Episodes of acute dehydroPA toxicity involving the 4.3. Congenital Anomalies 11 consumption of bread are avoided in many countries by 4.4. Liver Disease 11 more effective control of weeds in crops and by strictly applying 4.5. Exacerbating Factors 11 food safety standards limiting the number of foreign seeds in 5. Selected Indicators Suggesting a Possible Dehy- grain entering the human food chain, including some known to dropyrrolizidine Alkaloid Etiology 12 contain dehydroPAs. Flour millers also normally screen grain 6. Conclusions and Future Directions 12 Author Information 13 Received: July 31, 2014 Corresponding Author 13 Published: December 6, 2014

© 2014 American Chemical Society 4 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

Figure 1. Typical rural scenes showing plants producing dehydroPAs associated with agricultural production: (A) horses grazing a pasture infested with Senecio madagascariensis (fireweed) in Hawaii, (B) Jacobaea vulgaris (Senecio jacobaea, tansy ragwort) in a wheat crop in Germany, (C) Echium vulgare (blueweed, blue borage, viper’s bugloss) in a grain crop in Germany, and (D) a pasture infestation of Echium plantagineum (Paterson’s curse, Salvation Jane) in Australia. prior to milling to ensure that all foreign seeds and plant contaminated by dehydroPAs, e.g., up to 2000−4000 μgof fragments are removed. However, if dehydroPA-containing dehydroPAs/kg in some supermarket honeys.28,29,40 Levels of plants were present in the crop, then dust containing dehydroPAs up to 5647 μg/kg have recently been reported in dehydroPAs is not removed by screening and, consequently, teas purchased in retail markets in Germany.21 The levels of − some dehydroPAs remain associated with the grain.17 20 It is dehydroPAs found in these foods are generally too low to cause − normal practice in countries undertaking large-scale cereal acute hepatotoxicity.18,31,32,42 44 However, based on a consid- production to combine grain from many farms and from eration of their well-established genotoxicity8,45,46 and on different cropping areas. Such bulking dilutes and reduces the recent risk assessments suggesting that to make cancer unlikely level of unwanted contaminants in the grain, including a maximum tolerable daily intake of 0.007 μg of dehydroPAs/ − dehydroPAs, but it results in a wider distribution of any kg body weight (bw) should not be exceeded,31,42 44 the dehydroPAs that were present and thus increases the dehydroPAs in these foods could be contributing to somatic population exposed to low levels of these hazardous chemicals mutations, leading to a wide range of cancers.8,46 They may also in grain-based foods. While these measures and practices are be initiating other chronic diseases such as pulmonary arterial apparently sufficient to prevent the acute dehydroPA poisoning hypertension (PAH), leading to enlargement of the right that regularly occurs in countries in which such measures and ventricle and right heart congestive failure,8,20,47 and may be a − practices are not applied, it is still possible that occasional, low- cause of some congenital anomalies.8,20,47 49 level dietary exposure in grain-based products could be This Perspective provides a comprehensive review of the contributing worldwide to the incidence of several slowly characteristics of acute and chronic dehydroPA toxicity and developing, chronic diseases.18,19 suggests some associative considerations, or indicators, for Other food items increasingly being reported to be linking specific cases of cancer, PAH, congenital anomalies, and periodically contaminated by low levels of dehydroPAs include liver diseases with intermittent, low-level dietary exposure to milk, eggs, meat, honey, pollen, teas, salads, and foods dehydroPAs. Some of these indicators are then used to identify − containing these as ingredients.18,20 40 Both Echium spp. cases of chronic diseases reported in the literature that may shown in Figure 1, and a number of other dehydroPA- have resulted from dietary exposure to dehydroPAs. The producing plants, are widely used in commercial honey etiology of these cases warrants further investigation to confirm production,41 and the honey produced is significantly or negate this possibility.

5 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

Many cases of cancer, PAH, congenital anomalies, and liver monoesters (e.g., heliotrine), (b) open-chain diesters (e.g., disease are of unknown etiology, so establishing that they are lasiocarpine), (c) macrocyclic diesters (e.g., retrorsine), and (d) sometimes caused by dietary exposure to dehydroPAs should seco-alkaloids (e.g., senkirkine). This complexity is further lead to ways of reducing the incidence of these costly and amplified by their existence in plants as both free bases and as debilitating medical conditions. The challenge is to identify N-oxides (Figure 2), the latter predominating in many plant specific cases that have been initiated or exacerbated by dietary species.4,52 exposure to dehydroPAs. 2.2. Bioactivation and Mechanism of Toxicity. DehydroPA free bases are pro-toxins. Following ingestion, 2. TOXICOLOGY they are absorbed from the gut and converted in the liver, by cytochrome P450 monooxygenases (CYP450), specifically the 2.1. Occurrence, Evolution, and Chemistry. It has been fl CYP3A and CYP2B isoforms in humans, to 1-hydroxymethyl-7- estimated that 3% of all owering plants produce poisonous hydroxy-6,7-dihydropyrrolizine ester metabolites (DHP esters) 50 ff − dehydroPAs. They are very e ective insect-feeding deterrents (Figure 3).8,46,52 75 These “pyrrolic” metabolites are bifunc- and consequently have evolved independently on at least four 51 tional biological alkylating agents capable of causing tissue occasions in a number of different plant families. Approx- 52 4,52 damage and inducing genetic mutations. In vivo N-oxidation imately 500 potentially toxic dehydroPAs are known. of dehydroPAs by CYP450 enzymes and flavin-containing Varying in the character and/or stereochemistry of the necine mono-oxygenases68 is a detoxifying mechanism that leads to base (Figure 2) and the esterifying acids (necic acids), they can fi excretion of the water-soluble dehydroPAs N-oxides produced be classi ed into four main categories (Figure 2): (a) (Figure 3);52,76 however, a significant proportion of ingested dehydroPA N-oxides are reduced to their free base forms during passage through the gut and in the liver52 and thus, when they are present in food, they too contribute to hepatic formation of genotoxic, mutagenic, tissue-damaging DHP esters − (Figure 3).8,68,77 83 While the DHP ester metabolites, e.g., dehydroriddelliine shown in Figure 3 and dehydromonocrotaline (Figure 4), have − been chemically synthesized from dehydroPAs,84 87 they have never been isolated from in vivo or in vitro metabolic studies due to their extremely high chemical reactivity and very short half-lives, especially in aqueous environments.52,88 After formation in hepatocytes, the DHP ester metabolites react rapidly with nucleophilic functional groups (sulfhydryl, amino, hydroxyl) on DNA, proteins, and other substances, e.g., glutathione (GSH), in the liver to produce mixtures of C7 and C9 mono and di DHP adducts, the latter forming cross- links between −SH, −OH, and −NH groups on different molecules or on the same macromolecule (Figures 3 and − − 4).46,52,57 75,89 91 Rapid, spontaneous in vivo hydrolysis of the DHP esters produces the relatively more stable enantiomers of the DHP diol, (±)-1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine (DHP) (Figure 3), namely, dehydroheliotridine55 and dehy- droretronecine,92 or, more likely, a racemic mixture of these two enantiomers.52 Unlike the highly reactive DHP esters, the effects of which are largely if not entirely confined to the liver,8,52 DHP survives for a longer time in the body.52 It escapes from the liver and has been isolated and identified in both in vivo and in vitro studies of dehydroPA metabo- lism.54,55,92 While less chemically reactive than the precursor DHP esters, DHP retains significant bifunctional, biological alkylating potential, especially under mildly acid conditions, and, as with the DHP esters, it forms C7 and C9 DHP adducts − in vivo (Figure 3).52,87,93 97 DHP adducts have been detected in the liver, lung, kidney, blood, bone marrow, gastrointestinal tract, brain, muscles, pancreas, thymus, heart, spleen, and testes of animals exposed to dehydroPAs and following intra- − peritoneal or subcutaneous injection of DHP.4,25,52,90,98 103 DHP, the final and common toxic metabolite of all 104−106 107 fi dehydroPAs, is carcinogenic and immunosuppressive Figure 2. Three most common necine bases de ning dehydroPAs, viz., ff retronecine, heliotridine, and otonecine; a general structure represent- and mimics the antimitotic e ects of radiation in tissues 108−111 ing dehydroPA N-oxides; and structures representing the four main displaying high rates of mitosis. Circulating DHP is categories of dehydroPA pro-toxins. considered to be responsible for the effects of dehydroPAs

6 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

Figure 3. Liver metabolism of dehydroPAs, using the macrocyclic diester riddelliine as an example. outside of the liver. It is, however, highly water-soluble, and any 3. PATHOLOGICAL EFFECTS 52 DHP that remains unadducted is excreted (Figure 3). 3.1. Sinusoidal Obstruction Syndrome and Cirrhosis. The potential harm of dietary exposure to dehydroPAs does Following exposure to relatively high doses of dehydroPAs, e.g., not end with formation of the initial DHP adducts or excretion tens of milligrams of dehydroPAs per kilogram of bodyweight, of dehydroPA N-oxides and DHP. Mono DHP adducts, the liver sinusoidal endothelial cells have been shown, in animal retaining one active alkylating site at C7 or C9, remain models, to be the first to display the effects of the DHP ester potentially hazardous as alkylating agents in vivo.112 In accord metabolites produced in adjacent hepatocytes. They have been with this, a mixture of preformed galectin-1-DHP adducts observed to round-up, swell, and slough off, leading to blockage − (Figure 4), shown by mass spectrometry to be mainly of the microcirculation in the sinusoids.114 118 The resulting monoadducted, was found to be cytotoxic in human pulmonary hepatic sinusoidal obstruction syndrome (HSOS) (also referred artery endothelial cells.112 More importantly, it has been to as hepatic veno-occlusive disease or VOD) is highly 8,14 established that some DHP adducts, involving DHP attached to characteristic of acute dehydroPA poisoning seen in people fi weak nucleophiles, can dissociate and release DHP or transfer and leads to necrosis of surrounding liver tissue, brosis, 52,95−97,109 nodular hyperplasia, bile duct proliferation, and eventually to DHP to stronger nucleophilic sites. The in vivo 8,110,117−119 persistence of “a reservoir of stabilized pyrrolic alkylating agents cirrhosis and liver failure. DehydroPAs are the only (DHP adducts) which could subsequently interact with other naturally occurring agents in the environment known to produce HSOS when consumed, and they are frequently used tissue constituents”52 is one reason why the toxic effects of to produce animal models of clinical cases of HSOS and dehydroPAs continue to develop slowly over an extended chronic, progressive liver disease that are used to study the period even from a single exposure to these hazardous 52,95−97,109−111,113 development of, and potential treatments for, these dis- substances. The reversibility of the reaction eases.115,118,120,121 of dehydroPA metabolites with certain weak nucleophiles The particular susceptibility of the sinusoidal endothelial cells prolongs the effects and enhances the hazard of dietary is thought to be, in part, associated with increased activity of exposure to dehydroPAs relative to some other genotoxic matrix metalloproteinases (MMPs), e.g., MMP-9 (gelatinase substances, and this fact should be taken into account in B), that degrade the extracellular matrix, leading to the assessing the risk of foods contaminated by these alkaloids. endothelial cells being released.116,117,122 MMPs are normally

7 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

Figure 4. A typical mixture of DHP adducts produced by reaction of galectin-1 with dehydromonocrotaline.112

− suppressed by GSH. As sinusoidal GSH levels rapidly decrease experimental models of PAH.134 139 Doses of dehydroPAs due to alkylation by dehydroPA metabolites and the redox state required to produce PAH in experimental animals comparable of the sinusoids changes, the activity of the MMPs increases, to clinical cases of PAH in humans are generally lower than and HSOS develops. In support of this mechanism, it has been those required to produce HSOS. This may explain, therefore, demonstrated, for example, that GSH infused into the portal the occasional development of PAH, without notable liver vein prevents HSOS from developing in animals exposed to pathology, in people taking herbal medicines containing − dehydroPAs.116,123 dehydroPAs.140 142 While HSOS is considered to be highly indicative of Children form a significant cohort of PAH cases of unknown dehydroPA exposure8,111,119 and has developed in humans etiology. The PAH produced in nonhuman primates by consuming levels as low as tens of micrograms of dehydroPAs monocrotaline shows very similar pathology to PAH diagnosed − per kilogram of bodyweight per day,17,110,124,125 it is not always in children.130 132 Rodents given monocrotaline are apparently readily apparent. For example, when the liver damage develops a less appropriate model of the human disease,131,132,136 but the over an extended period, due to relatively low-level exposure to monocrotaline-rodent model has become the most widely used dehydroPAs, HSOS may not be apparent or may be obscured to study the development of PAH and to test treatments for by hepatic fibrosis and nodular regeneration.110 Under these this condition.137,138 Stenmark et al.136 and Gomez-Arroyo et circumstances, the resulting cirrhosis may be indistinguishable al.139 have discussed some of the differences between the from cirrhosis caused by other agents, e.g., alcohol, aflatoxins, monocrotaline-rodent models of PAH and PAH seen in and hepatitis viruses, and the etiology can be ambiguous.110,119 humans. These differences appear to relate to the initial acute Thus, as well as clinical cases of HSOS, some cases of cirrhosis effects of dehydroPAs that are not seen in clinical cases of PAH. of unknown cause and without obvious HSOS could also have a These discordant effects could be explained, in part, by the dietary dehydroPA etiology. rodent model of PAH typically being generated using a single, 3.2. Pulmonary Arterial Hypertension (PAH). Recog- relatively high dose of monocrotaline, sufficient to produce nition that ingestion of dehydroPAs causes PAH leading to PAH relatively quickly in a high proportion of animals and not enlargement of the right ventricle and right heart failure (cor just the most susceptible. A long-term, intermittent, lower level pulmonale) was established many years ago in experimental of exposure, similar to that expected from the levels of − animals,56,126 133 and, since then, dehydroPAs such as dehydroPAs currently being detected in food, may avoid the monocrotaline and fulvine have been deliberately administered early discordant acute effects sometimes seen in the standard by medical researchers to various animal species to generate monocrotaline-rodent model of PAH136,139 and may be

8 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective expected to slowly produce PAH more typical of the disease dehydroPA N-oxide, indicine-N-oxide, was at one time clinically normally seen in humans and in the nonhuman primate model investigated for use as a cancer chemotherapeutic agent.148,149 − developed by Chesney and Allen.130 132 The antimitotic effect of dehydroPAs exposure can, therefore, Several experiments with nonhuman primates provide be expected to initially inhibit both cancer development and the insights into what can be expected from long-term, low-level development of the quasi-malignant endothelial cells in the − dietary exposure to dehydroPAs.130 132 A highly significant, pulmonary arterioles that characterize PAH.150 The develop- age-related difference was observed following subcutaneous ment of both cancer and PAH will, therefore, be particularly injection of the relatively high dose of 30 mg/kg bw favored by extended, low-level, and intermittent periods of monocrotaline, followed by three further injections, 2 months dehydroPA exposure, as is expected from current knowledge of apart, of 60 mg/kg bw, to 4 week old and 15 month old foods contaminated by dehydroPAs, as this type of exposure monkeys.130 The older animals, as expected from the very high allows intervening periods of normal mitotic activity that provide opportunity for cancers and progressive PAH to doses administered, developed HSOS and showed typical − dehydroPA liver pathology, whereas the young monkeys develop.8,52,110,150 155 showed minimal liver pathology but developed PAH typical 3.4. Teratogenicity. Exposure of pregnant women to of that seen in children. It was established that the young dehydroPAs in herbal teas, herbal medicines, and spices has monkeys displayed much lower cytochrome CYP450 activity in caused fatal HSOS in neonates.8,52,110,125 DehydroPAs and their livers and would therefore have produced insufficient their DHP metabolites are teratogenic in rats, and it has been levels of the monocrotaline DHP ester metabolite (dehydro- shown that they cross the placenta and form DHP adducts in monocrotaline) to rapidly deplete sinusoidal GSH, activate rat embryos.48,49,52 Growth retardation, abnormal skeletal MMPs, release sinusoidal endothelial cells, and cause HSOS. development, and deficiencies in ossification are common They must, however, have produced sufficient low levels of features. Congenital anomalies have, however, not yet been circulating, labile DHP adducts and DHP to damage the extensively studied or considered as a possible consequence of endothelium of the pulmonary arteries and cause PAH. The low-level dietary exposure to dehydroPAs. Nonetheless, as well response of the younger monkeys represents the situation that as stillbirths and HSOS, congenital anomalies are to be would most likely pertain if a human diet regularly delivered expected in surviving neonates following dietary exposure of very small amounts of dehydroPAs that yielded insufficient their mothers to dehydroPAs during pregnancy. toxic metabolites to cause HSOS but sufficient to initiate PAH and perhaps cause somatic mutations leading to other 4. POSSIBLE INDICATORS OF genotoxic conditions such as cancer. Thus, PAH and cancer, DEHYDROPYRROLIZIDINE ALKALOID rather than HSOS, are the most likely outcomes from INVOLVEMENT circulating low microgram per kilogram of bodyweight levels Growing evidence of significant dehydroPA contamination of a of labile DHP adducts and free DHP. This would be especially wide range of common foods suggests that they could be a so if dietary exposure to dehydroPAs is intermittent since this common cause of several chronic diseases.20 The ability of would allow the well-established antimitotic effects of dehydroPAs to cause disparate chronic diseases, viz., cancers, dehydroPA metabolites to be relatively short-lived and enable PAH, HSOS, and genetic anomalies, suggests that disease cancers and progressive PAH, both requiring cell proliferation, complexes involving these diseases could be used to indicate to develop. This type of intermittent, low-level dietary exposure the involvement of dehydroPAs. This section considers the seems increasingly likely to have occurred in the past and to identification of diseases potentially caused by dietary continue to the present day to varying degrees in many regions dehydroPAs in this context. throughout the world.20,47 Current knowledge of the relatively 4.1. Cancer. Linking a diverse range of cancers with somatic low but significant levels of dehydroPAs in food and an mutations arising from relatively constant or, more effectively, expectation of intermittent dietary exposure, insufficient to intermittent, low-level, long-term dietary exposure to dehy- produce HSOS, suggest that dietary dehydroPAs could be droPAs is likely to be difficult, but the highly characteristic responsible for at least some cases of clinical PAH of currently ability of dehydroPAs to produce HSOS, PAH, and genetic unknown etiology reported in the medical literature. anomalies could be used as clinical signs suggesting such a link. 3.3. Carcinogenesis. It has been shown, using exper- Evidence of one or more of these other conditions can be imental animals and human cell cultures, that one of several expected in at least some patients with cancers caused by genetic targets for toxic dehydroPA metabolites is the gene dehydroPAs. TP53, encoding the tumor suppressor protein p53.46,143,144 As well as exposure to dehydroPAs, another clinically Signature mutations of dehydroPAs include G:C → T:A recognized and common cause of dehydroPA-like HSOS is transversion and tandem base substitutions.46 Cancers of the associated with chemotherapy of childhood cancers such as liver, lung, kidney, skin, intestines, bladder, brain and spinal rhabdomyosarcoma (RMS), nephroblastoma (Wilms tumor), cord, pancreas, adrenal gland, muscle (rhabdomyosarcoma, metastatic colorectal cancer, and leukemia using alkylating − RMS), and blood (leukemia) have been produced by agents such as cyclophosphamide or busulfan.156 164 Treat- dehydroPAs and their metabolites in experimental ani- ment of hematologic cancers in neonates by allogenic stem cell mals.8,46,145,146 These are also the tissues in which DHP transplantation (SCT) following myeloablation using these − adducts have been detected. A very similar, wide range of agents can also cause HSOS as a complication.164 177 cancers is associated with Li-Fraumeni syndrome caused by Like dehydroPAs, cyclophosphamide and busulfan are germline mutations of TP53,147 a known target of dehydroPA biological alkylating agents and thus may simply mimic − metabolites. dehydroPAs in causing HSOS,178 180 although, unlike As previously mentioned, dehydroPAs and their DHP dehydroPAs, neither of these agents is used to produce animal metabolites display strong antimitotic effects8,52,106 typical of models of HSOS. Indeed, cyclophosphamide is not generally many other biological alkylating agents used to treat cancer. A considered to be hepatotoxic,181 so it is somewhat unexpected

9 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective that it causes HSOS in some childhood cancer patients. It is, PAH.185 The dehydroPA monocrotaline and its DHP ester however, possible that if the cancer being treated developed as metabolite (dehydromonocrotaline, “monocrotaline pyrrole”, a consequence of dietary exposure to dehydroPAs, for example Figure 4) cause mutation of BMP signaling genes in animal − when fetuses are exposed to dehydroPA metabolites in utero or models of PAH,185 187 and their ability to cause mutations of as infants consuming dehydroPA-contaminated breast milk BMP-signaling genes is clearly a significant factor in the ability and/or dehydroPA-contaminated food, then asymptomatic of dehydroPAs to cause PAH. HSOS may co-occur with their cancers. Overt HSOS can A hallmark feature of both clinical PAH and dehydroPA- then be precipitated by the additional insult to the sinusoids induced PAH are the proliferative, quasi-malignant, hyper- from chemotherapeutic agents not normally capable of causing trophic (megalocytic), apoptosis-resistant cells, in particular HSOS. Dietary exposure to low levels of dehydroPAs may be, smooth muscle cells, that contribute to the characteristic like radiotherapy, a priming event for HSOS when cancers plexiform (onionskin-like) lesions responsible for progressive caused by dehydroPAs are treated with therapeutic alkylating vascular remodeling and vasoconstriction of the pulmonary − agents such as cyclophosphamide and busulfan. arterioles.138,188 194 The previously mentioned susceptibility of DehydroPAs have been shown to induce biosynthesis of muscle cells to DHP alkylation leading to RMS may have a GSH in the liver in response to the depletion of GSH they parallel in the development of the quasi-malignant smooth cause.182 The potential genotoxicity associated with continued muscle cells in the pathogenesis of PAH. dietary exposure to dehydroPAs during induction of GSH In regard to the hypertrophic character of the pulmonary biosynthesis could cause mutations in the upregulated GSH artery endothelial cells involved in PAH, similar long-lived, biosynthesis genes. In turn, this could lead to compromise of morphologically and functionally viable megalocytes have long the ability of some dehydroPA-affected livers to biosynthesize been recognized as a highly characteristic aberration of − GSH and thus dehydroPA-exposed livers could become hepatocytes in dehydroPA-poisoned animals.4,52,195 198 They sensitized to other alkylating agents such as cyclophosphamide have also been described in the lungs,152 kidney,4,152 and or busulfan that normally do not cause HSOS. In this regard, it duodenum111 of animals exposed to dehydroPAs. While would be of interest to compare the development of HSOS in functionally viable and long-lived, the hepatic megalocytes experimental animals using cyclophosphamide and busulfan that develop in surviving animals following acute dehydroPA- with and without priming by dietary exposure to very low levels poisoning initially appear to be incapable of division.196,197 This of dehydroPAs and also to investigate whether the GSH characteristic is, however, believed to be due to the persistent biosynthesis capability of livers is compromised by such antimitotic effects of dehydroPAs, and, given time, some of priming. these hypertrophic cells are expected to “escape from mitotic Rhabdomyosarcoma (RMS) is a cancer originating from inhibition.”197 In some cases, the proliferative, hypertrophic, mutations in muscle progenitor cells.183 DHP becomes a more apoptosis-resistant smooth muscle cells, seen in the pulmonary aggressive alkylating agent under mildly acid conditions due to arterioles of patients with PAH, could arise in this way and be protonation of the alcohol groups, providing a good leaving indicative of intermittent, low-level dietary exposure to entity (H2O) for generating the carbonium ions involved in dehydroPAs. alkylation.52,87,95 Therefore, accumulation of lactic acid in Twenty percent of idiopathic PAH cases are associated with muscle tissue could quite conceivably result in myoblasts being BMP receptor II (BMPR2) mutations, and 75% of familial PAH a favored site for DHP alkylation, causing mutations and cases display germline mutations of BMPR2.199,200 These − resulting in RMS. In accord with this possibility, while mutations alone are, however, insufficient to cause PAH.199 201 intraperitoneal injection of DHP into rats produces a range The low penetrance (20%) of PAH among carriers of BMPR2 of cancers similar to those produced by ingestion of the parent mutations has led to the suggestion that additional environ- dehydroPAs,106 subcutaneous injection of the DHP enantiomer mental factors and further somatic mutations may be needed dehydroretronecine produced RMS at the site of injection in before PAH develops in people carrying BMPR2 mutations. It 65% of rats (39 of 60).104,105 A dehydroPA etiology for some has therefore been suggested that a so-called second hit is cases of RMS is, therefore, in accord with this observation and required before PAH develops.200,201 Dietary exposure to also with HSOS being a complication in a significant dehydroPAs could deliver such a second hit in carriers of proportion of RMS patients when they undergo chemotherapy BMPR2 mutations and lead to the development of PAH in involving alkylating agents such as cyclophosphamide and these individuals. Thus, dietary exposure to dehydroPAs may busulfan. not only be responsible for the development of PAH in Cancer clusters involving several types of cancer could also previously mutation-negative individuals but also could be an be indicative of a dietary dehydroPA origin. Such heteroge- important contributor to the development of PAH in carriers of neous clusters are often dismissed as unlikely to have a BMPR2 mutations. common etiology. However, Daughton184 has drawn attention Portopulmonary hypertension (POPH) is a relatively rare to the co-occurrence of acute lymphoblastic leukemia and RMS form of PAH associated with cirrhosis and portal hyper- − in Nevada and Arizona and has suggested that this coincidence tension.202 204 The exact pathogenesis and etiology of POPH could indicate a dietary dehydroPA etiology. It would be of are poorly understood.205 One clinical study of 2459 patients interest to determine if there was concurrently or subsequently with cirrhosis showed that 15 (0.61%) exhibited POPH and an increased incidence of cirrhosis, HSOS, and PAH in those concluded that an unknown dietary cause was “the most communities. attractive explanation for the association of portal and 4.2. Pulmonary Arterial Hypertension. Dysregulation of pulmonary hypertension.”206 Another study207 has suggested bone morphogenetic protein (BMP) signaling is a feature of that the “prevalence of primary pulmonary hypertension clinical cases of both sporadic (idiopathic) and inherited associated with cirrhosis may be greater than previously (familial) PAH and has been implicated in the vascular cell reported.” The combination of cirrhosis, portal hypertension, proliferation and remodeling of pulmonary arterioles seen in and PAH, characteristic of POPH, resembles the chronic

10 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective condition sometimes caused by dehydroPAs in animals, so Environmental copper is known to exacerbate the dietary dehydroPAs could conceivably be, in some cases, a hepatotoxicity of dehydroPAs in livestock and to result in differential consideration in determining the etiology of POPH- accumulation of very high levels of copper in the cirrhotic − like diseases. liver.4,52,77,227 230 The poisoning of livestock by dehydroPAs All cases of PAH of uncertain etiology need to be was at one time called chronic copper toxicity until it was investigated as being possibly caused, initiated, or exacerbated shown that this particular condition also required exposure to by dietary exposure to dehydroPAs. plants containing dehydroPAs.4,227 Numerous investigations 4.3. Congenital Anomalies. The skeletal defects seen in have sought to explain individual examples and clusters of rat fetuses whose mothers were exposed to heliotrine or cirrhosis in young children that have occurred in Germany, DHP48,49 strongly suggest that the alkaloids are causing Austria, India, the USA, Australia, and elsewhere. Such cases are 222−226,231−242 deficiencies in BMP signaling.208,209 As previously discussed, sometimes associated with copper accumulation. mutation of BMP signaling genes by dehydroPA metabolites Many investigations have concluded, however, that high levels 235,237,238 contributes to their ability to cause PAH in experimental of environmental copper are not the only cause. Some animals, so it is not surprising that ineffective bone formation is have suggested that other environmental agents, including 240,241 among the congenital anomalies seen in animals exposed to exposure to dehydroPAs and perhaps a non-Wilsonian 48,49 genetic predisposition to copper accumulation and toxicity, may dehydroPA metabolites in utero. 234,236−240,242−244 HSOS is commonly experienced as a complication of be required. This situation is very reminiscent of the historic debate in the 1950s on the etiology of chronic conditioning regimes involving cyclophosphamide and busulfan 4,52,227 used for prior marrow ablation when, for example, congenital copper toxicity in livestock. Cirrhosis accompanied by anomalies such as malignant infantile osteopetrosis (MIOP), copper accumulation is, therefore, another important indication primary immunodeficiency (PID), and severe immune for suspecting possible dietary dehydroPA involvement. dysregulatory disorders (SIDs) are treated by SCT.210,211 If A form of HSOS called hepatic veno-occlusive disease with immunodeficiency (VODI) is associated with germline these congenital anomalies developed as a consequence of 245−250 maternal exposure to dehydroPAs, then such exposure could mutations of the SP110 nuclear body protein gene. SP110 nuclear body protein is involved in regulation of the prime the livers of the neonates for development of HSOS ’ during myeloablation. body s immune response, so a nonfunctional version of SP110 As well as displaying a predisposition to developing HSOS nuclear body protein will lead to impairment of the immune system.251 It is, however, less clear how the loss of SP110 following SCT, some MIOP patients also frequently develop 252 severe PAH as a complication.210 PAH was also the most nuclear body protein can cause HSOS. This suggests a possible role for another factor such as dietary dehydroPAs. common cause of death in a study of 48 patients with The known immunotoxicity of dehydroPAs and DHP107,108,253 fibrodysplasia ossification progressiva (FOP).212 Of the 48 FOP could conceivably contribute to the development of a VODI- patients studied, 26 (54%) died prematurely (age range 8−58, like disease in susceptible individuals with or without a pre- median life span 42 years) from PAH. Another possible existing germline mutation of the SP110 gene. It is even indication of a dehydroPA etiology is that FOP is characterized possible that mutation of the SP110 gene by dehydroPA by congenital toe malformations and ossification issues 213,214 metabolites could contribute to or account for some of the involving BMP signaling abnormalities. immune system impairment associated with dehydroPA Beckwith−Wiedemann syndrome (BWS), some features of 107 215,216 exposure. which involve faulty BMP signaling, is another congenital 4.5. Exacerbating Factors. Modern bulking of commod- disorder that could sometimes have a dehydroPA etiology. ities such as grain, milk, and honey for wide national and BWS is associated with significantly increased risk of tumors 217−219 international distribution leads to dilution of dehydroPA such as RMS, hepatoblastoma, and Wilms tumor, contaminants and ensures that dietary exposure to acutely including, in some cases, the co-occurrence of RMS and the hazardous levels of dehydroPAs is relatively rare in the many anaplastic subtype of Wilms tumor associated with mutations of countries where such bulking normally occurs. Thus, hazardous 220,221 fi the TP53 gene. The de ciencies in BMP signaling as well levels of dietary exposure to dehydroPAs are likely to occur as the susceptibility to developing certain cancers, including more commonly in particular localities and communities or RMS, and concurrent susceptibility to HSOS during chemo- households due to specific items of food produced and therapy could quite conceivably indicate that the congenital consumed locally being contaminated by dehydroPAs at certain defects of BWS, the cancers that develop, and the susceptibility times. Regional and/or familial clusters of dehydroPA- to HSOS may sometimes all have a common dehydroPA origin. associated chronic diseases are particularly likely in areas In combination, these observations suggest that a dehydroPA where locally produced and consumed honey, milk, eggs, grain, etiology should be considered as a possible cause of some cases and other foods are occasionally contaminated via dehydroPA- of BWS, MIOP, FOP, PID, and SID and possibly other producing plants that grow in that region and especially during congenital conditions. times when incidents of livestock poisoning are being 4.4. Liver Disease. Dietary exposure to dehydroPAs should reported.254 A familial genetic propensity for hepatic activation also be considered for all cases of cirrhosis of unknown of dehydroPAs, rather than detoxication, associated with etiology, especially those showing evidence of HSOS. While inherited CYP450 profiles can also be expected to result in clinicians dealing with cases of HSOS, especially in neonates family clusters of dehydroPA-initiated chronic diseases. and young children, often question the mothers of the patients Some medications, e.g., rifampicin, St. Johns wort, and involved in regard to their consumption of herbal medicines phenobarbital, and environmental contaminants, e.g., poly- − containing dehydroPAs,222 226 the potential for dehydroPAs chlorinated biphenyls, induce cytochrome CYP3A enzymes, so being present in foods they have consumed, such as milk, concurrent consumption of or exposure to these could increase honey, flour, tea, and eggs, is not normally considered. an individual’s susceptibility to dehydroPA toxicity via

11 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective enhanced bioactivation. Viruses, bacterial endotoxins, and (HSOS),238 and rare mitotic figures223 that are indicative of a aflatoxins have also been shown to exacerbate liver damage dehydroPA etiology. and cancers caused by dehydroPAs.255,256 If several of the indicators suggested above are present in an The particular susceptibility of fetuses and nursing neonates individual case, then the possibility that dietary exposure to to toxic effects when their mothers consume foods or herbal dehydroPAs is involved deserves further investigation, 8,20,257,258 medicines containing dehydroPAs is well-established. especially if other features of dehydroPA poisoning are also Fetal and neonate liver CYP450 enzymes are generally less present such as hypertrophic (megalocytic) cells, antimitotic ff 52,130 e ective in activating dehydroPA, and it is thought that the effects, and evidence of immunotoxicity. DHP metabolites produced by and transferred from the mother are largely responsible for the harm caused to fetuses and breast 52 6. CONCLUSIONS AND FUTURE DIRECTIONS feeding neonates. In recognition of the susceptibility of fetuses and nursing neonates, German regulations for Recent recognition that some individuals and communities may phytopharmaceuticals specify that dehydroPA-containing herb- be intermittently exposed to hazardous levels of mutagenic al medicines cannot be sold to pregnant or breast feeding dehydroPAs, naturally present or present as contaminants in a 257,258 women. For other consumers in Germany, the daily dose number of common foods, raises the possibility that these of dehydroPAs in a small number of specified dehydroPA- natural toxins may be important contributors to the develop- μ containing phytopharmaceuticals must not exceed 1 g, ment of certain chronic diseases in those individuals and μ reduced to 0.1 g if the medication is taken daily for more communities. However, epidemiological support is required, than 6 weeks per year.257 These regulations are not currently fi and considerable research is needed to establish the veracity applied to food, although a signi cant number of retail food and extent of this possibility. As well as increased regional items have been shown to exceed the maximum levels of analyses of food to determine current levels of dietary exposure dehydroPAs allowed in phytopharmaceuticals in Ger- − − to dehydroPAs, awareness among clinicians of this possibility many.21,27 29,33 40 should lead to questioning of patients about their dietary habits 5. SELECTED INDICATORS SUGGESTING A POSSIBLE and the sources of the foods they consume. DEHYDROPYRROLIZIDINE ALKALOID ETIOLOGY The chronic diseases that could sometimes be caused, initiated, or precipitated by the current intermittent, low levels It will be necessary to eventually develop criteria, well of dietary exposure to dehydroPAs include (a) a wide range of supported by targeted research and epidemiological studies, cancers, (b) PAH, (c) progressive liver disease leading to which can be used to assist in identifying diseases that may be associated with dietary exposure to dehydroPAs. Until this is cirrhosis, and (d) congenital anomalies, or a combination of achieved, the following indicators, rationally based on these. PAH and HSOS, in particular, are highly characteristic associative considerations described above, are recommended and indicative responses to dehydroPA exposure. All cases of as possible indications of a dietary dehydroPA etiology and chronic illness involving or accompanied by evidence of either suggest the need for further investigations into possible sources or both of these relatively rarely co-occurring conditions could 160,207 of exposure: indicate a possible dietary dehydroPA etiology. The availability of methods for detecting and quantifying (1) Latent or overt HSOS and PAH of unknown or − 71,261 − 262 uncertain etiology. DHP DNA and DHP protein adducts provides means of measuring levels of dietary exposure to dehydroPAs (2) Cirrhosis, especially if associated with HSOS and/or in different populations and allows the level of DHP adducts accumulation of copper in the liver. detected to be correlated with the incidence of certain chronic (3) Cancers and/or congenital anomalies where there is diseases. Such analysis is, for example, particularly called for in evidence of overt or asymptomatic HSOS, PAH, bone fi regions where honeys from dehydroPA-producing plants are deformities, or immunological de ciencies. harvested and sold locally or when flour from locally harvested (4) PAH accompanied by evidence of hepatotoxicity. grain could sometimes be contaminated. These data can then (5) Any of the above occurring in consanguineous or be correlated with the relative incidence of associated chronic regional clusters, especially in areas where dehydroPA- diseases in these particular regions. producing weeds grow and where locally produced The current animal models of PAH produced by exposure to dehydroPA-contaminated foods are likely to occasionally dehydroPAs are aimed at generating high numbers of affected occur and especially if there is concurrent evidence of animals relatively quickly for experimental purposes, and some livestock being poisoned by dehydroPAs.254 acute effects of dehydroPAs are, therefore, also produced. (6) Any of the above involving fetuses, neonates, and While these animal models do indeed reflect all of the children. important aspects of the pathophysiology of PAH, they do not In considering and selecting the six indicators comprising the above list, several cases of liver disease and cancers with a mimic likely low level and intermittent dietary exposure to possible dehydroPA etiology have been identi- these natural toxins. There is a need to explore these animal − − − − − fied.158 160,163,168,176,222 226,231 234,236 238,245,247 249,259,260 Of models using levels of dehydroPAs equivalent to those expected − these, ten cases159,160,176,223,237,238,245,247 249 display at least from current levels and patterns of food contamination in at- four of the suggested indicators, nine risk communities. Congenital anomalies in fetuses and others158,163,222,224,225,232,233,258,260 have three indicators, and neonates, caused by dehydroPA at levels equivalent to those five other cases168,226,231,234,236 have two of the suggested currently occurring in the food supply, also need to be explored indicators. Some of these cases also showed other features, in animal models, as does the apparent role of defective BMP including hypertrophic hepatocytes (ballooned, swel- signaling as a common mechanism in, and cause of, several ling),222,232,233,237,238 inconsistent veno-occlusive disease possible dehydroPA-associated diseases.

12 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective ■ AUTHOR INFORMATION (15) Kleiman, R., Rentz, E. D., Teshale, E., Thompson, N., and Schurz-Rogers, H. (2008) Update on research and activities at the Corresponding Author Centers for Disease Control and Prevention, and the Agency for Toxic *Tel: +61 2 9490 5000; Fax: +61 2 9490 8499; E-mail: john. Substances and Disease Registry. J. Med. Toxicol. 4, 197−200. [email protected]. (16) Kakar, F., Akbarian, Z., Leslie, T., Mustafa, M. L., Watson, J., van Notes Egmond, H. P., Omar, M. F., and Mofleh, J. (2010) An outbreak of fi hepatic veno-occlusive disease in western Afghanistan associated with The authors declare no competing nancial interest. exposure to wheat flour contaminated with pyrrolizidine alkaloids. J. Toxicol. 2010, 313280. ■ ABBREVIATIONS (17) Edgar, J. A. (2003) Pyrrolizidine alkaloids and food safety. Chem. Aust. 70,4−7. HSOS, hepatic sinusoidal obstruction syndrome; dehydroPAs, (18) Food Standards Australia New Zealand (FSANZ) (2001) 1,2-dehydropyrrolizidine ester alkaloids; PAH, pulmonary Pyrrolizidine alkaloids in food. A toxicological review and risk assessment, arterial hypertension; bw, body weight; CYP450, cytochrome Tech. Report series No. 2, FSANZ, Canberra, Australia, http://www. P450 monoamine oxidases; DHP, 1-hydroxymethyl-7-hydroxy- foodstandards.gov.au/publications/documents/TR2.pdf. 6,7-dihydropyrrolizine; GSH, glutathione; MMPs, metallopro- (19) Azadbakht, M., and Talavaki, M. (2003) Qualitative and teinases; POPH, portopulmonary hypertension; BMPR2, bone quantitative determination of pyrrolizidine alkaloids of wheat and flour morphogenic protein receptor II; VODI, hepatic veno-occlusive contaminated with Senecio in Mazandaran Province farms. Iran. J. disease with immunodeficiency; RMS, rhabdomyosarcoma; Pharm. Res. 2, 179−183. ́ SCT, allogeneic stem cell transplantation; MIOP, malignant (20) Edgar, J. A., Colegate, S. M., Boppre, M., and Molyneux, R. J. fi (2011) Pyrrolizidine alkaloids in food: a spectrum of potential health infantile osteopetrosis; PID, primary immunode ciency; SIDs, − severe immune dysregulatory disorders; FOP, fibrodysplasia consequences. Food Addit. Contam. 28, 308 324. fi (21) Bodi, D., Ronczka, S., Gottschalk, C., Behr, N., Skibba, A., ossi cation progressive; BWS, Beckwith-Wiedemann Syndrome Wagner, M., Lahrssen-Wiederholt, M., Preiss-Weigert, A., and These, A. (2014) Determination of pyrrolizidine alkaloids in tea, herbal drugs ■ REFERENCES and honey. Food Addit. Contam. 31, 1886−1895. (1) Willmot, F. C., and Robertson, G. W. (1920) Senecio disease, or (22) Dickinson, J. O., and King, R. R. (1978) The transfer of cirrhosis of the liver due to Senecio poisoning. Lancet, 848−849. pyrrolizidine alkaloids from Senecio jacobaea into milk of lactating cows and goats, in Effects of Poisonous Plants on Livestock (Keeler, I., (2) Steyn, D. G. (1933) Poisoning of human beings by weeds − contained in cereals (Bread Poisoning). Onderstepoort J. Vet. Sci. Anim. VanKampen, K. R., and James, L. F., Eds.) pp 201 206, Academic Ind. 1, 219−266. Press, New York. (3) Selzer, G., and Parker, R. G. F. (1951) Senecio poisoning as (23) Deinzer, M. L., Thomson, P. A., Burgett, D. M., and Isaacson, D. Chiari’s syndrome. A report on twelve cases. Am. J. Pathol. 27, 885− L. (1977) Pyrrolizidine alkaloids: their occurrence in honey from tansy − 907. ragwort (Senecio jacobaea L.). Science 195, 497 499. (4) Bull, L. B., Culvenor, C. C. J., and Dick, A. J. (1968) The (24) Culvenor, C. C. J., Edgar, J. A., and Smith, L. W. (1981) Pyrrolizidine Alkaloids. Their Chemistry, Pathogenicity and Other Pyrrolizidine alkaloids in honey from Echium plantagineum L. J. Agric. − Biological Properties, North Holland Publishing Co., Amsterdam, The Food Chem. 29, 958 960. (25) Lüthy, J., Heim, T., and Schlatter, C. (1983) Transfer of Netherlands. 3 (5) Tandon, B. N., Tandon, R. K., Tandon, H. D., Narndranathan, [ H]pyrrolizidine alkaloids from Senecio vulgaris L. and metabolites − M., and Joshi, Y. K. (1976) An epidemic of veno-occlusive disease of into rat milk and tissues. Toxicol. Lett. 17, 283 288. liver in central India. Lancet 2, 271−272. (26) Molyneux, R. J., and James, L. F. (1990) Pyrrolizidine alkaloids (6) Tandon, R. K., Tandon, B. N., Tandon, H. D., Bhatia, M. L., in milk: thresholds of in toxication. Vet. Hum. Toxicol. 32 (Suppl.), − Bhargava, S., Lal, P., and Arora, R. R. (1976) Study of an epidemic of 94S 103S. venoocclusive disease in India. Gut 17, 849−855. (27) Edgar, J. A., and Smith, L. W. (2000) Transfer of pyrrolizidine (7) Mohabbat, O., Younos, M. S., Merzad, A. A., Srivastava, R. N., alkaloids into eggs: food safety implications, in Natural and Selected ffi Sediq, G. G., and Aram, G. N. (1976) An outbreak of hepatic veno- Synthetic Toxins, Biological Implications. (Tu, A. T., and Ga eld, W., occlusive disease in North-Western Afghanistan. Lancet 308, 269−271. Eds.) ACS Symposium Series 745, Chapter 8, pp 118−128, American (8) International Programme on Chemical Safety (IPCS) (1988) Chemical Society, Washington, DC. Pyrrolizidine alkaloids: Environmental Health Criteria 80,WHO, (28) Beales, K. A., Betteridge, K., Colegate, S. M., and Edgar, J. A. Geneva, Switzerland, www.inchem.org/documents/ehc/ehc/ehc080. (2004) Solid phase extraction and LCMS analysis of pyrrolizidine htm. alkaloids in honeys. J. Agric. Food Chem. 52, 6664−6672. (9) International Programme on Chemical Safety (IPCS) (1989) (29) Beales, K, Betteridge, K, Boppre,́ M, Cao, Y, Colegate, S. M., Pyrrolizidine alkaloids: health and safety guide no. 26; WHO, Geneva, Edgar, J. A. (2007) Hepatotoxic pyrrolizidine alkaloids and their N- Switzerland, http://www.inchem.org/documents/hsg/hsg/hsg026. oxides in honey and pollen, in Poisonous Plants: Global Research and htm. Solutions (Panter, K., Wierenga, T., and Pfister, J., Eds.) Chapter 16, pp (10) Chauvin, P., Dillon, J., Moren, A., Talbak, S., and Barakaev, S. 94−100, CABI, Wallingford. (1993) Heliotrope poisoning in Tadjikistan. Lancet 341, 1663. (30) Bundesamt für Risiokobewertung (BfR) (2007) Salatmischung (11) Chauvin, P., Dillon, J. C., and Moren, A. (1994) An outbreak of mit pyrrolizidinalkaloid-haltigem Greiskraut verunreinigt, Stellungnahme heliotrope food poisoning, Tadjikistan, November 1992−March 1993. Nr.028/2007 des BfR vom 10, http://www.bfr.bund.de/cm/208/ Sante 4, 263−268. salatmischung_mit_pyrrolizidinalkaloid_haltigem_geiskraut_ (12) Mayer, F., and Lüthy, J. (1993) Heliotrope poisoning in verunreinigt.pdf. Tadjikistan. Lancet 342, 246−247. (31) Bundesamt für Risiokobewertung (BfR) (2011) Chemical (13) Altaee, M. Y., and Mahmood, M. H. (1998) An outbreak of analysis and toxicity of pyrrolizidine alkaloids and assessment of the veno-occlusive disease of the liver in northern Iraq. East. Mediterr. health risk posed by their occurrence in honey, BfR opinion no. 038/2011 Health J. 4, 142−148. 11, http://www.bfr.bund.de/cm/349/chemical-analysis-and-toxicity- (14) Integrated Regional Information Networks (IRIN) (2008) of-pyrrolizidine-alkaloids-and-assessment-of-the-health-risks-posed-by- Afghanistan: “Charmak” disease still killing people, livestock in west,UN their-occurence-in-honey.pdf. Office for the Coordination of Humanitarian Affairs, http://www. (32) Bundesamt für Risiokobewertung (BfR) (2013) Pyrrolizidine irinnews.org/Report.aspx?ReportId=81971. alkaloids in herbal teas and teas, BfR opinion no. 018/2013, http://

13 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective www.bfr.bund.de/cm/349/pyrrolizidine-alkaloids-in-herbal-teas-and- defense system in separate angiosperm lineages. Plant Cell 16, 2772− teas.pdf. 2784. (33) Kempf, M., Heil, S., Hasslauer, I., Schmidt, L., von der Ohe, K., (52) Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine Theuring, C., Reinhard, A., Scheier, P., and Beuerle, T. (2010) Alkaloids, Academic Press, London, UK. Pyrrolizidine alkaloids in pollen and pollen products. Mol. Nutr. Food (53) Mattocks, A. R. (1968) Toxicity of pyrrolizidine alkaloids. Res. 54,1−9. Nature 217, 723−728. (34) Hoogenboom, L. A. P., Mulder, P. P. J., Zeilmaker, M. J., van (54) Culvenor, C. C. J., Downing, D. T., Edgar, J. A., and Jago, M. V. den Top, H. J., Remmelink, G. J., Brandon, E. F. A., Klijnstra, M., (1969) Pyrrolizidine alkaloids as alkylating and antimitotic agents. Meijer, G. A. L., Schothorst, R., and van Egmond, H. P. (2011) Carry- Ann. N.Y. Acad. Sci. 163, 837−847. over of pyrrolizidine alkaloids from feed in dairy cows. Food Addit. (55) Jago, M. V., Edgar, J. A., Smith, L. W., and Culvenor, C. C. J. Contam. 28, 359−372. (1970) Metabolic conversion of heliotridine-based pyrrolizidine (35) Dübecke, A., Beckh, G., and Lüllmann, C. (2011) Pyrrolizidine alkaloids to dehydroheliotridine. Mol. Pharmacol. 6, 402−406. alkaloids in honey and bee pollen. Food Addit. Contam. 28, 348−358. (56) Butler, W. H., Mattocks, A. R., and Barnes, J. M. (1970) Lesions (36) Fletcher, M. T., McKenzie, R. A., Reichmann, K. G., and Blaney, in the liver and lungs of rats given pyrrole derivatives of pyrrolizidine − B. J. (2011) Risks from plants containing pyrrolizidine alkaloids for alkaloids. J. Pathol. 100, 169 175. livestock and meat quality in northern Australia, in Poisonings by Plants, (57) Williams, D. E., Reed, R. L., Kedzierski, B., Dannan, G. A., Mycotoxins and Related Toxins (Riet-Correa, F., Pfister, J., Schild, A. L., Guengerich, F. P., and Buhler, D. R. (1989) Bioactivation and and Wierenga, T., Eds.) pp 208−218, CAB International, Wallingford, detoxication of the pyrrolizidine alkaloid senecionine by cytochrome − UK. P-450 enzymes in rat liver. Drug Metab. Dispos. 17, 387 392. (37) Kempf, M., Wittig, M., Schönfeld, K., Cramer, L., Schreier, P., (58) Miranda, C. L., Reed, R. L., Guengerich, F. P., and Buhler, D. R. and Beuerle, T. (2011) Pyrrolizidine alkaloids in food: downstream (1991) Role of cytochrome P450IIIA4 in the metabolism of the pyrrolizidine alkaloid senecionine in human liver. Carcinogenesis 12, contamination in the food chain caused by honey and pollen. Food − Addit. Contam. 28, 325−331. 515 519. (38) Kempf, M., Wittig, M., Reinhard, A., von der Ohe, K., (59) Hincks, J. R., Kim, H.-Y., Segall, H. J., Molyneux, R. J., Stermitz, Blacquiere,́ T., Raezke, K.-P., Michel, R., Scheier, P., and Beuerle, T. F. R., and Coulombe, R. A. (1991) DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure−activity relationships. (2011) Pyrrolizidine alkaloids in honey: comparison of analytical − methods. Food Addit. Contam. 28, 332−347. Toxicol. Appl. Pharmacol. 111,90 98. (39) Cao, Y., Colegate, S. M., and Edgar, J. A. (2013) Persistence of (60) Chung, W. G., and Buhler, D. R. (1994) The effect of spironolactone treatment on the cytochrom P450-mediated metabo- echimidine, a hepatotoxic pyrrolizidine alkaloid, from honey into lism of the pyrrolizidine alkaloid senecionine by hepatic microsomes mead. J. Food Compos. Anal. 29, 106−109. from rats and guinea pigs. Toxicol. Appl. Pharmacol. 127, 314−319. (40) Griffin, C. T., Danaher, M., Elliott, C. T., Kennedy, D. G., and (61) Chung, W. G., Miranda, C. L., and Buhler, D. R. (1995) A Furey, A. (2013) Detection of pyrrolizidine alkaloids in commercial cytochrome P450B form is the major bioactivation enzyme for the honey using liquid chromatography-ion trap mass spectrometry. Food pyrrolizidine alkaloid senecionine in guinea pig. Xenobiotica 25, 929− Chem. 136, 1577−1583. 939. (41) Edgar, J. A., Roeder, E., and Molyneux, R. J. (2002) Honey from (62) Kim, H.-Y., Stermitz, F. R., and Coulombe, R. A. (1995) plants containing pyrrolizidine alkaloids: a potential threat to health. J. − Pyrrolizidine alkaloid-induced DNA-protein cross-links. Carcinogenesis Agric. Food Chem. 50, 2719 2730. 16, 2691−2697. (42) Committee on Toxicity of Chemicals in Food, Consumer (63) Kasahara, Y., Kiyatake, K., Tatsumi, K., Sugito, K., Kakusaka, I., Products and the Environment (COT) (2008) COT statement on Yamagata, S., Ohmori, S., Kitada, M., and Kuriyama, T. (1997) pyrrolizidine alkaloids in food, http://cot.food.gov.uk/pdfs/ Bioactivation of monocrotaline by P450 3A in rat liver. J. Cardiovasc. cotstatementpa200806.pdf. Pharmacol. 30, 124−129. (43) WHO (2011) Discussion paper on pyrrolizidine alkaloids, Joint (64) Reid, M. J., Lame,́ M. W., Morin, D., Wilson, D. W., and Segall, FAO/WHO food standards programme, Codex committee on H. J. (1998) Involvement of cytochrome P450 3A in the metabolism contaminants in foods, 5th session, The Hague, The Netherlands, and covalent binding of 14C-monocrotaline in rat liver microsomes. J. − 21 25 March, ftp://ftp.fao.org/codex/meetings/cccf/cccf5/cf05_14e. Biochem. Mol. Toxicol. 12, 157−166. pdf. (65) Pereira, T. N., Webb, R. I., Reilly, P. E. B., Seawright, A. A., and (44) European Food Safety Authority (EFSA) Panel on Prakash, A. S. (1998) Dehydromonocrotaline generates sequence- Contaminants in the Food Chain (CONTAM) (2011) Scientific selective N-7 guanine alkylation and heat and alkali stable multiple − opinion on pyrrolizidine alkaloids in food and feed. EFSA J. 9, 2406 fragment DNA crosslinks. Nucl. Acid Res. 26, 5441−5447. 2540. (66) Tepe, J. J., and Williams, R. M. (1999) DNA cross-linking by a (45) The U.S. National Toxicology Program (2011) 12th Report on phototriggered dehydromonocrotaline progenitor. J. Am. Chem. Soc. Carcinogens (RoC), pp iii−499, U.S. Department of Health and Human 121, 2951−2955. Services, Washington, DC. (67) Lin, G., Cui, Y. Y., and Hawes, E. M. (2000) Characterization of (46) Chen, T., Mei, N., and Fu, P. P. (2010) Genotoxicity of rat liver microsomal metabolites of clivorine, an hepatotoxic pyrrolizidine alkaloids. J. Appl. Toxicol. 30, 183−196. otonecine-type pyrrolizidine alkaloid. Drug Metab. Dispos. 28, 1475− (47) Edgar, J. A. (2014) Food contaminants capable of causing 1483. cancer, pulmonary hypertension and cirrhosis. Med. J. Aust. 200,73− (68) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2004) Pyrrolizidine 74. alkaloidsgenotoxicity, metabolism enzymes, metabolic activation, (48) Green, C. R., and Christie, G. S. (1961) Malformation in foetal and mechanisms. Drug Metab. Rev. 36,1−55. rats induced by the pyrrolizidine alkaloid, heliotrine. Br. J. Exp. Pathol. (69) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2002) Genotoxic 42, 369−378. pyrrolizidine alkaloidsmechanism leading to DNA adduct formation (49) Peterson, J. E., and Jago, M. V. (1980) Comparison of the toxic and tumorigenicity. Int. J. Mol. Sci. 3, 948−964. effects of dehydroheliotridine and heliotrine in pregnant rats and their (70) Yang, Y.-C., Yan, J., Doerge, D. R., Chan, P.-C., Fu, P. P., and embryos. J. Pathol. 131, 339−355. Chou, M. W. (2001) Metabolic activation of the tumorigenic (50) Smith, L. W., and Culvenor, C. C. J. (1981) Plant sources of pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation hepatotoxic pyrrolizidine alkaloids. J. Nat. Prod. 44, 129−152. in vivo. Chem. Res. Toxicol. 14, 101−109. (51) Reimann, A., Nurhayati, N., Backenköhler, A., and Ober, D. (71) Yang, Y.-C., Yan, J., Churchwell, M., Beger, R., Chan, P.-C., (2004) Repeated evolution of the pyrrolizidine alkaloid-mediated Doerge, D. R., Fu, P. P., and Chou, M. W. (2001) Development of a

14 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

32P-postlabeling/HPLC method for detection of dehydroretronecine- excretion, and transfer into milk of lactating mice. Drug Metab. Dispos. derived DNA adducts in vivo and in vitro. Chem. Res. Toxicol. 14,91− 10, 236−240. 100. (91) Chou, M. W., and Fu, P. P. (2006) Formation of DHP-derived (72) Xia, Q., Chou, M. W., Kadlubar, F. F., Chan, P., and Fu, P. P. DNA adducts in vivo from dietary supplements and Chinese herbal (2003) Human liver microsomal metabolism and DNA adduct plant extracts containing carcinogenic pyrrolizidine alkaloids. Toxicol. formation of the tumorigenic pyrrolizidine alkaloid, riddelliine. Ind. Health 22, 321−327. Chem. Res. Toxicol. 16,66−73. (92) Hsu, I. C., Allen, J. R., and Chesney, C. F. (1973) Identification (73) Chou, M. W., Yan, J., Nichols, J., Xia, Q., Beland, F. A., Chan, P. and toxicological effects of dehydroretronecinemetabolite of C., and Fu, P. P. (2003) Correlation of DNA adduct formation and monocrotaline. Proc. Soc. Exp. Biol. Med. 144, 834−838. riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice. (93) Hsu, I. C., Robertson, K. A., Shumaker, R. C., and Allen, J. R. Cancer Lett. 193, 119−125. (1975) Binding of tritiated dehydroretronecine to macromolecules. (74) Xia, Q., Chou, M. W., Lin, G., and Fu, P. P. (2004) Metabolic Res. Commun. Chem. Pathol. Pharmacol. 11,99−106. formation of DHP-derived DNA adducts from a representative (94) Curtain, C. C., and Edgar, J. A. (1976) The binding of otonecine type pyrrolizidine alkaloid clivorine and the extract of dehydroheliotridine to DNA and the effect of it and other compounds Ligularia hodgsonnii Hook. Chem. Res. Toxicol. 17, 702−708. on repair synthesis in main and satellite band DNA. Chem.−Biol. (75) Xia, Q., Chou, M. W., Edgar, J. A., Doerge, D. R., and Fu, P. P. Interact. 13, 243−256. (2006) Formation of DHP-derived DNA adducts from metabolic (95) Sun, P. S., Hsia, M-T. S., Chu, F. S., and Allen, J. R. (1977) activation of the prototype heliotridine-type pyrrolizidine alkaloid, Inhibition of yeast alcohol dehydrogenase by dehydroretronecine. lasiocarpine. Cancer Lett. 231, 138−145. Food Cosmet. Toxicol. 15, 419−422. (76) Mattocks, A. R., and Bird, I. (1983) Pyrrolic and N-oxide (96) Seawright, A. A. (1992) Potential toxicity problems with herbal metabolites formed from pyrrolizidine alkaloids by hepatic micro- medicines and foodnew observations with pyrrolizidine alkaloids. somes in vitrorelevance to in vivo hepatotoxicity. Chem.−Biol. Proc. Nutr. Soc. Aust. 17,20−25. Interact. 43, 209−222. (97) Seawright, A. A. (1994) Toxic plant residues in meat, in Plant- (77) Bull, L. B., and Dick, A. T. (1959) The chronic pathological Associated Toxins; Agricultural, Phytochemical and Ecological Aspects effects on the liver of the rat of the pyrrolizidine alkaloids heliotrine, (Colegate, S. M., and Dorling, P. R., Eds.) Chapter 15, pp 77−84, CAB lasiocarpine and their N-oxides. J. Pathol. Bacteriol. 78, 483−502. International, Wallingford UK. (78) Mattocks, A. R. (1971) Hepatotoxic effects due to pyrrolizidine (98) Hsu, I. C., Schumaker, R. C., and Allen, J. R. (1974) Tissue alkaloid N-oxides. Xenobiotica 1, 563−565. distribution of tritium-labeled dehydroretronecine. Chem.−Biol. (79) Molyneux, R. J., Johnson, A. E., Olson, J. D., and Baker, D. C. Interact. 8, 163−170. (1991) Toxicity of pyrrolizidine alkaloids from Riddell groundsel (99) Hsu, I. C., Robertson, K. A., and Allen, J. R. (1976) Tissue (Senecio riddellii) to cattle. Am. J. Vet. Res. 52, 146−151. distribution, binding properties and lesions produced by dehydrore- (80) Chou, M. W., Wang, J. Y., Yang, Y.-C., Beger, R. D., Williams, L. tronecine in the non-human primate. Chem.−Biol. Interact. 12,19−28. D., Doerge, D. R., and Fu, P. P. (2003) Riddelliine N-oxide is a (100) Shumaker, R. C., Hsu, I. C., and Allen, J. R. (1976) phytochemical and mammalian metabolite with genotoxic activity that Localisation and tissue effects of tritiated dehydroretronecine in young is comparable to the parent pyrrolizidine alkaloid riddelliine. Toxicol. rats. J. Pathol. 119,21−28. Lett. 145, 239−247. (101) Mattocks, A. R., and White, I. N. H. (1976) The distribution of (81) Wang, Y.-P., Yan, J., Fu, P. P., and Chou, M. W. (2005) Human [3H]synthanecine a bis-N-ethylcarbamate and its metabolites in the liver microsomal reduction of pyrrolizidine alkaloid N-oxides to form rat. Chem.−Biol. Interact. 15, 173−184. the corresponding carcinogenic parent alkaloid. Toxicol. Lett. 155, (102) Candrian, U., Lüthy, J., and Schlatter, C. (1985) In vivo 411−420. covalent binding of retronecine-labeled [3H] seneciphylline and [3H] (82) Yan, J., Xia, Q., Chou, M. W., and Fu, P. P. (2008) Metabolic senecionine to DNA of rat liver, lung and kidney. Chem.−Biol. Interact. activation of retronecine and retronecine-N-oxideformation of 54,57−69. DHP-derived DNA adducts. Toxicol. Ind. Health 24, 181−188. (103) Mattocks, A. R., and Jukes, R. (1990) Recovery of the pyrrolic (83) Cao, Y., Colegate, S. M., and Edgar, J. A. (2008) Safety nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates assessment of food and herbal products containing hepatotoxic in tissues and body fluids. Chem.−Biol. Interact. 75, 225−239. pyrrolizidine alkaloids: inter-laboratory consistency and the impor- (104) Allen, J. R., Hsu, I.-C., and Carstens, L. A. (1975) tance of N-oxide determination. Phytochem. Anal. 19, 526−533. Dehydroretronecine induced rhabdomyosarcomas in rats. Cancer Res. (84) Mattocks, A. R. (1968) Iron (II)-catalysed conversion of 35, 997−1002. unsaturated pyrrolizidine alkaloid N-oxides to pyrrole derivatives. (105) Shumaker, R. C., Robertson, K. A., Hsu, I. C., and Allen, J. R. Nature 219, 480. (1976) Neoplastic transformation in tissues of rats exposed to (85) Culvenor, C. C. J., Edgar, J. A., Smith, L. W., and Tweedale, H. J. monocrotaline or dehydroretronecine. J. Natl. Cancer Inst. 56, 787− (1969) Dihydropyrrolizine analogues of pyrrolizidine alkaloids. Aust. J. 789. Chem. 41, 3599−3602. (106) Peterson, J. E., Jago, M. V., Reddy, J. K., and Jarrett, R. G. (86) Mattocks, A. R. (1969) Dihydropyrrolizine derivatives from (1983) Neoplasia and chronic disease associated with the prolonged unsaturated pyrrolizidine alkaloids. J. Chem. Soc. C, 1155−1162. administration of dehydroheliotridine to rats. J. Natl. Cancer Inst. 70, (87) Culvenor, C. C. J., Edgar, J. A., Smith, L. W., and Tweedale, H. J. 381−386. (1970) Dihydropyrrolizines III. Preparation and reactions of (107) Percy, J., and Pierce, A. E. (1971) Immunosuppressive activity derivatives related to pyrrolizidine alkaloids. Aust. J. Chem. 23, of the pyrrolizidine alkaloid metabolite dehydroheliotridine. Immunol- 1853−1867. ogy 21, 273−280. (88)Mattocks,A.R.,andJukes,R.(1990)Trappingand (108) Peterson, J. E., Samuel, A., and Jago, M. V. (1972) Pathological measurement of short-lived alkylating agents in a recirculating flow effects of dehydroheliotridine, a metabolite of heliotridine-based system. Chem.−Biol. Interact. 76,19−30. pyrrolizidine alkaloids in the young rat. J. Pathol. 107, 175−189. (89) Mattocks, A. R., Driver, H. E., Barbour, R. H., and Robins, D. J. (109) Mattocks, A. R., and Bird, I. (1983) Alkylation by (1986) Metabolism and toxicity of synthetic analogues of macrocyclic dehydroretronecine, a cytotoxic metabolite of some pyrrolizidine diester pyrrolizidine alkaloids. Chem.−Biol. Interact. 58,95−108. alkaloids: an in vivo test. Toxicol. Lett. 16,1−8. (90) Eastman, D. F., Dimenna, G. P., and Segall, H. J. (1982) (110) Prakash, A. S., Pereira, T. N., Reilly, P. E. B., and Seawright, A. Covalent binding of two pyrrolizidine alkaloids, senecionine and A. (1999) Pyrrolizidine alkaloids in human diet. Mutat. Res. 443,53− seneciphylline, to hepatic macromolecules and their distribution, 67.

15 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

(111) Hooper, P. T. (1975) Experimental acute gastrointestinal non-human primates (Macaca arctoides). Am. J. Vet. Res. 34, 1577− disease caused by the pyrrolizidine alkaloid, lasiocarpine. J. Comp. 1581. Pathol. 85, 341−349. (132) Chesney, C. F., and Allen, J. R. (1973) Monocrotaline induced (112) Wong, L. S.N., Lame,́ M. W., Jones, A. D., and Wilson, D. W. pulmonary vascular lesions in non-human primates. Cardiovasc. Res. 7, (2010) Differential cellular responses to protein adducts of 508−518. naphthoquinone and monocrotaline pyrrole. Chem. Res. Toxicol. 23, (133) Fishman, A. P. (1974) Dietary pulmonary hypertension. Circ. 1504−1513. Res. 35, 657−660. (113) Molyneux, R. J., Johnson, A. E., and Stuart, L. D. (1988) (134) Pan, L. C., Wilson, D. W., Lame,́ M. W., Jones, A. D., and Delayed manifestation of Senecio-induced pyrrolizidine alkaloidosis in Segall, H. J. (1993) Cor pulmonale is caused by monocrotaline and cattle: case reports. Vet. Hum. Toxicol. 30, 201−205. dehydromonocrotaline, but not by glutathione or cysteine conjugates (114) DeLeve, L. D., Wang, X., Kuhlenkamp, J. F., and Kaplowitz, N. of dihydropyrrolizine. Toxicol. Appl. Pharmacol. 118,87−97. (1996) Toxicity of azathioprine and monocrotaline in murine (135) Sehgal, P. B., and Mukhopadhyay, S. (2007) Dysfunctional sinusoidal endothelial cells and hepatocytes: the role of glutathione intracellular trafficking in the pathobiology of pulmonary arterial and relevance to hepatic venooclusive disease. Hepatology 23, 589− hypertension. Am. J. Respir. Cell Mol. Biol. 37,31−37. 599. (136) Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., and (115) DeLeve, L. D., McCuskey, R. S., Wang, X., Hu, L., McCuskey, McMurtry, I. F. (2009) Animal models of pulmonary arterial M. K., Epstein, R. B., and Kanel, G. C. (1999) Characterization of a hypertension: the hope for etiological discovery and pharmacological reproducible rat model of hepatic veno-occlusive disease. Hepatology cure. Am. J. Physiol.: Lung Cell. Mol. Physiol. 297, L1013−1032. 29, 1779−1791. (137) Yen, C. H., Tsai, T. H., Leu, S., Chen, Y. L., Chang, L. T., Chai, (116) DeLeve, L. D., Shulman, H. M., and McDonald, G. B. (2002) H. T., Chung, S. Y., Chua, S., Tsai, C. Y., Chang, H. W., Ko, S. F., Sun, Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome C. K., and Yip, H. K. (2013) Sildenafil improves long term effect of (veno-occlusive disease). Semin. Liver Dis. 22,27−41. endothelial progenitor cell-based treatment for monocrotaline-induced (117) DeLeve, L. D., Ito, Y., Bethea, N. W., McCuskey, M. K., Wang, rat pulmonary arterial hypertension. Cytotherapy 15, 209−223. X., and McCuskey, R. S. (2003) Embolization by sinusoidal lining cells (138) Gupta, V., Gupta, N., Shaik, I. H., Mehvar, R., Nozik-Grayck, obstructs the microcirculation in rat sinusoidal obstruction syndrome. E., McMurtry, I. F., Oka, M., Komatsu, M., and Ahsan, F. (2013) − Am. J. Physiol.: Gastrointest. Liver Physiol. 284, G1045 G1052. Inhaled PLGA particles of prostaglandin E1 ameliorate symptoms and (118) DeLeve, L. D., Valla, D.-C., and Garcia-Tsao, G. (2009) progression of pulmonary hypertension at a reduced dosing frequency. Vascular disorders of the liver. Hepatology 49, 1729−1764. Mol. Pharmaceutics 10, 1655−1667. (119) Huxtable, R. J. (1989) Human health implications of (139) Gomez-Arroyo, J. G., Farkas, L., Alhussaini, A. A., Farkas, D., pyrrolizidine alkaloids and herbs containing them, in Toxicants of Kraskauskas, D., Voelkel, N. F., and Bogaard, H. J. (2012) The Plant Origin (Cheeke, P. R., Ed). Vol. 1, Chapter 3, pp 42−86, CRC monocrotaline model of pulmonary hypertension in perspective. Am. J. Press, Inc., Boca Raton, FL. Physiol.: Lung Cell. Mol. Physiol. 302, L363−369. (120) Nolan, J. P., Scheig, R. L., and Klatskin, G. (1966) Delayed (140) Kay, J. M., Heath, D., Smith, P., Bras, G., and Summerell, J. hepatitis and cirrhosis in weanling rats following a single small dose of (1971) Fulvine and pulmonary circulation. Thorax 26, 249−261. the Senecio alkaloid, lasiocarpine. Am. J. Pathol. 49, 129−151. (141) Heath, D., Shaba, J., Williams, A., Smith, P., and Kombe, A. (121) Gordon, G. J., Coleman, W. B., and Grisham, J. W. (2000) (1975) A pulmonary hypertension-producing plant from Tanzania. Temporal analysis of hepatocyte differentiation by small hepatocyte- Thorax 30, 399−404. like progenitor cells during liver regeneration in retrorsine-exposed (142) Györika, S., and Stricker, H. (2009) Severe pulmonary rats. Am. J. Pathol. 157, 771−786. hypertension possibly due to pyrrolizidine alkaloids in polyphytother- (122) Hanumegowda, U. M., Copple, B. L., Shibuya, M., Malle, E., apy. Swiss Med. Wkly. 139, 210−211. Ganey, P. E., and Roth, R. A. (2003) Basement membrane and matrix (143) Couet, C. E., Hanley, A. B., MacDonald, S., and Mayes, L. metalloproteinases in monocrotaline-induced liver injury. Toxicol. Sci. (1993) The biological assay of natural mutagens using p53, in Food 76, 237−246. and Cancer Prevention (Waldron, K. W., Johnston, I. T., and Fenwick, (123) Wang, X., Kanel, G. C., and DeLeve, L. D. (2000) Support of G. R., Eds.) pp 426−428, Royal Society of Chemistry, London. sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive (144) Ji, L., Zhang, M., Sheng, Y., and Wang, Z. (2005) Pyrrolizidine disease in the rat. Hepatology 31, 428−434. alkaloid clivorine induces apoptosis in human normal liver L-20 cells (124) Ridker, P. M., Ohkuma, S., McDermott, W. V., Trey, C., and and reduces the expression of p53 protein. Toxicol. In Vitro 19,41−46. Huxtable, R. J. (1985) Hepatic venooclusive disease associated with (145) Hirono, I. (1993) Edible plants containing naturally occurring the consumption of pyrrolizidine-containing dietary supplements. carcinogens in Japan. Jpn. J. Cancer Res. 84, 997−1006. Gastroenterology 88, 1050−1054. (146) Chan, P. C., Haseman, J. K., Prejean, J. D., and Nyska, A. (125) Rasenack, R., Müller, C., Kleinschmidt, M., Rasenack, J., and (2003) Toxicity and carcinogenicity of riddelliine in rats and mice. Wiedenfeld, H. (2003) Veno-occlusive disease in a fetus caused by Toxicol. Lett. 144, 295−311. pyrrolizidine alkaloids of food origin. Fetal Diagn. Ther. 18, 223−225. (147) Malkin, D. (2011) Li-Fraumeni syndrome. Genes Cancer 2, (126) Kay, J. M., and Heath, D. (1966) Observations on the 475−484. pulmonary arteries and heart weight of rats fed on Crotalaria spectabilis (148) LeTendre, L., Ludwig, J., Perrault, J., Smithson, W. A., and seeds. J. Pathol. Bacteriol. 92, 385−394. Kovach, J. S. (1984) Hepatocellular toxicity during treatment of (127) Heath, D., and Kay, J. M. (1967) Medial thickness of refractory acute leukemia with indicine N-oxide. Cancer 54, 1256− pulmonary trunk in rats with cor pulmonale induced by ingestion of 1259. Crotalaria spectabilis seeds. Cardiovasc. Res. 1,74−79. (149) Miser, J. S., Smithson, W. A., Krivit, W., Hughes, C., Davis, D., (128) Kay, J. M., Harris, P., and Heath, D. (1967) Pulmonary Krailo, M., and Hammond, D. (1991) Phase II trial of indicine N-oxide hypertension produced in rats by ingestion of Crotalaria spectabilis in relapsed pediatric solid tumors. Invest. New Drugs 9, 339−342. seeds. Thorax 22, 176−179. (150) Rai, P. R., Cool, C. D., King, J. A. C., Stevens, T., Burns, N., (129) Burns, J. (1972) The heart and pulmonary arteries in rats fed Winn, R. A., Kasper, M., and Voelkel, N. F. (2008) The cancer on Senecio jacobaea. J. Pathol. 106, 187−194. paradigm of severe pulmonary arterial hypertension. Am. J. Respir. Crit. (130) Allen, J. R., and Chesney, C. F. (1972) Effect of age on Care Med. 178, 558−564. development of cor pulmonale in non-human primates following (151) Schoental, R. (1968) Toxicology and carcinogenic action of pyrrolizidine alkaloid intoxication. Exp. Mol. Pathol. 17, 220−232. pyrrolizidine alkaloids. Cancer Res. 28, 2237−2246. (131) Chesney, C. F., and Allen, J. R. (1973) Endocardial fibrosis (152) McLean, E. K. (1970) The toxic actions of pyrrolizidine associated with monocrotaline induced pulmonary hypertension in (Senecio) alkaloids. Pharmacol. Rev. 22, 429−483.

16 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

(153) Svoboda, D. J., and Reddy, J. K. (1972) Malignant tumors in (170) Barker, C. C., Butzner, J. D., Anderson, R. A., Brant, R., and rats given lasiocarpine. Cancer Res. 32, 908−912. Sauve, R. S. (2003) Incidence, survival and risk factors for the (154) Hirono, I., Shimizu, M., Fushimi, K., Mori, H., and Kato, K. development of veno-occlusive disease in pediatric hematopoietic stem (1973) Carcinogenic activity of Petasites japonicus Maxim, a kind of cell transplant recipients. Bone Marrow Transplant. 32,79−87. coltsfoot. Gann Monogr. Cancer Res. 64, 527−528. (171) Kraemer, D. M., Waschke, J., and Kunzmann, V. (2003) (155) Hirono, I., Mori, H., and Haga, M. (1978) Carcinogenic Venoocclusive disease in a male patient with Marfam syndrome and activity of Symphytum officinale. J. Natl. Cancer Inst. 61, 865−869. common acute lymphoblastic leukemia during induction therapy. Ann. (156) Kanwar, V. S., Albuquerque, M. L., Ribeiro, R. C., Kauffman, Hematol. 82, 444−447. W. M., and Furman, W. L. (1995) Veno-occlusive disease of the liver (172) Bajwa, R. P. S., Cant, A. J., Abinun, M., Flood, T. J., Hodges, S., after chemotherapy for rhabdomyosarcoma: case report with a review Hale, J. P., and Skinner, R. (2003) Recombinant tissue plasminogen of the literature. Med. Pediatr. Oncol. 24, 334−340. activator for treatment of hepatic veno-occlusive disease following (157) Ortega, J. A., Donaldson, S. S., Ivy, S. P., Pappo, A., and bone marrow transplantation in children: effectiveness and a scoring Maurer, H. M. (1997) Venoocclusive disease of the liver after system for initiating treatment. Bone Marrow Transplant. 31, 591−597. chemotherapy with vincristine, actinomycin D, and cyclophosphamide (173) Kalayoglu-Besisik, S., Yenerel, M. N., Caliskan, Y., Ozturk, S., for the treatment of rhabdomyosarcoma. Cancer 79, 2435−2439. Besisik, F., and Sargin, D. (2005) Time-related changes in the (158) Bisogno, G., de Kraker, J., Weirich, A., Masiero, L., Ludwig, R., incidence severity and clinical outcome of hepatic veno-occlusive Tornade, M. F., and Carli, M. (1997) Veno-occlusive disease of the disease in hematopoietic stem cell transplantation patients during the liver in children treated for Wilms tumor. Med. Pediatr. Oncol. 29, past ten years. Transplant. Proc. 37, 2285−2289. 245−251. (174) Papadopoulos, A., Ntaios, G., Kaiafa, G., Girtovitis, Z. S., (159) Ludwig, R., Weirich, A., Abel, U., Hofmann, W., Graf, N., and Kontoninas, Z., Diamantidis, M. D., Savopoulos, C., and Hatzitolios, A. Tournade, M. F. (1999) Hepatotoxicity in patients treated according (2008) Veno-occlusive disease of the liver during induction therapy for to the nephroblastoma trial and study SIOP-9/GPOH. Med. Pediatr. acute lymphoblastic leukemia. Int. J. Hematol. 88, 441−442. Oncol. 33, 462−469. (175) Dulley, F. L., Kanfer, E. J., Appelbaum, F. R., Amos, D., Hill, R. (160) Burkhardt, A., and Klöppel, A. (1977) Unusual obliterative S., Buckner, C. D., Shulman, H. M., McDonald, G. B., and Thomas, E. disease of the hepatic veins in an infant. Virchows Arch. A: Pathol. Anat. D. (1987) Veno-occlusive disease of the liver after chemoradiotherapy Histol. 375, 225−232. and autologous bone marrow transplantation. Transplantation 43, (161) Rubbia-Brandt, L., Audard, V., Sartoretti, P., Roth, A. D., 870−873. Brezault, C., Le Charpentier, M., Dousset, B., Morel, P., Soubrane, O., (176) Carreras, E. (2000) Veno-occlusive disease of the liver after Chausade, S., Mentha, G., and Terris, B. (2004) Severe hepatic hemopoietic cell transplantation. Eur. J. Haematol. 64, 281−291. sinusoidal obstruction associated with oxaliplatin-based chemotherapy (177) Kumar, S., DeLeve, L. D., Kamath, P. S., and Tefferi, A. (2003) in patients with metastatic colorectal cancer. Ann. Oncol. 15, 460−466. Hepatic veno-occlusive disease (sinusoidal obstruction syndrome) (162) Boula, A. M., Mantadakis, E., Xilouri, I. M., Christoforidou, A. after hematopoietic stem cell transplantation. Mayo Clin. Proc. 78, V., Foudoulakis, A. M., and Samonis, G. (2005) Veno-occlusive disease 589−598. of the liver associated with chronic myelomonocytic leukemia treated (178) Gharib, M. I., Bulley, S. R., Doyle, J. J., and Wynn, R. F. (2006) with vincristine and standard doses of cytarabine. Am. J. Hematol. 79, Venous occlusive disease in children. Thromb. Res. 118,27−38. 216−219. (179) Rubbia-Brandt, L., Tauzin, S., Brezault, C., Delucinge-Vivier, (163) Elli, M., Pinarli, F. G., Dagdemir, A., and Acar, S. (2006) Veno- C., Descombes, P., Dousset, P. E., Mentha, G., and Terris, B. (2011) occlusive disease of the liver in a child after chemotherapy for brain Gene expression profiling provides insights into pathways of tumor. Pediatr. Blood Cancer 46, 521−523. oxaliplatin-related sinusoidal obstruction syndrome in humans. Mol. (164) Matute-Bello, G., McDonald, G. D., Hinds, M. S., Schoch, H. Cancer Ther. 10, 687−696. G., and Crawford, S. W. (1998) Association of pulmonary function (180) De Jonge, M. E., Huitema, A. D. R., Beijnen, J. H., and testing abnormalities and severe veno-occlusive disease of the liver Rodenhuis, S. (2006) High exposures to bioactivated cyclophospha- after marrow transplantation. Bone Marrow Transplant. 21, 1152− mide are related to the occurrence of veno-occlusive disease of the 1130. liver following high-dose chemotherapy. Br. J. Cancer 94, 1226−1230. (165) McDonald, G. B., Hinds, M. S., Fisher, L. D., Schoch, H. G., (181) Plapp, F. V., and Chiga, M. (1972) Disaggregation of mouse Wolford, J. L., Banaji, M., Hardin, B. J., Shulman, H. M., and Clift, R. liver polysomes by cyclophosphamide. Arch. Pharmacol 272, 351−353. A. (1993) Veno-occlusive disease of the liver and multiorgan failure (182) Yan, C. C., and Huxtable, R. J. (1996) Effects of after bone marrow transplantation: a cohort study of 355 patients. Ann. monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in Int. Med. 118, 255−267. the rat. Biochem. Pharmacol. 51, 375−379. (166) Strasser, S. I., Sullivan, K. M., Myerson, D., Spurgeon, C. L., (183) Hettmer, S., and Wagers, A. J. (2010) Uncovering the origins Storer, B., Schoch, H. G., Murakami, C. S., and McDonald, G. B. of rhabdomyosarcoma. Nat. Med. 16, 171−173. (1999) Cirrhosis of the liver in long-term marrow transplant survivors. (184) Daughton, C. G. (2005) Overlooked in Fallon? Environ. Health Blood 93, 3259−3266. Perspect. 113, A224. (167) Copelan, E. A., Penza, S. L., Elder, P. J., Ezzone, S. A., Scholl, (185) Morty, R. E., Nejman, B., Kwapiszewska, G., Hecker, M., M. D., Bechtel, T. P., Belt, P. S., and Avalos, B. R. (2000) Analysis of Zakrzewicz, A., Kouri, F. M., Peters, D. M., Dumitrascu, R., Seeger, W., prognostic factors for allogenic marrow transplantation following Knaus, P., Schermuly, R. T., and Eickelberg, O. (2007) Dysregulated busulfan and cyclophosphamide in myelodysplastic syndrome and bone morphogenetic Protein signaling in monocrotaline-induced after leukemic transformation. Bone Marrow Transplant. 25, 1219− pulmonary arterial hypertension. Arterioscler., Thromb., Vasc. Biol. 27, 1222. 1072−1078. (168) Horn, B., Reiss, U., Matthay, K., McMillan, A., and Cowan, M. (186) Murakami, K., Mathew, R., Huang, J., Farahani, R., Peng, H., (2002) Veno-occlusive disease of the liver in children with solid Olson, S. C., and Etlinger, J. D. (2010) Smurf1 ubiquitin ligase causes tumors undergoing autologous hematopoietic progentitor cell trans- downregulation of BMP receptors and is induced in monocrotaline plantation: a high incidence in patients with neuroblastoma. Bone and hypoxia models of pulmonary arterial hypertension. Exp. Biol. Marrow Transplant. 29, 409−415. Med. 235, 805−813. (169) Bairey, O., Kirgner, I., Yakobi, M., Hamdan, A., Ben-Ari, Z., (187) Ramos, M., Lame,́ M. W., Segall, H. J., and Wilson, D. W. and Shaklai, M. (2002) Clinical severe hepatic venooclusive disease (2007) Monocrotaline pyrrole induces Smad nuclear accumulation during induction treatment of acute monoblastic leukemia managed and altered signaling expression in human pulmonary arterial with defibrotide. Am. J. Hematol. 69, 281−284. endothelial cells. Vasc. Pharmacol. 46, 439−448.

17 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

(188) Owens, G. K., Rabinovitch, P. S., and Schwartz, S. M. (1981) (207) Murata, K., Shimizo, A., Takase, K., Nakano, T., and Tameda, Smooth muscle hypertrophy versus hyperplasia in hypertension. Proc. Y. (1997) Asymptomatic primary pulmonary hypertension associated Natl. Acad. Sci. U.S.A. 78, 7759−7763. with liver cirrhosis. J. Gastroenterol. 32, 102−104. (189) Veyssier-Belot, C., and Cacoub, P. (1999) Role of endothelial (208) Bragdon, B., Moseychuk, O., Saldanha, S., King, D., Julian, J., and smooth muscle cells in the physiopathology and treatment and Nohe, A. (2011) Bone morphogenetic proteins: a critical review. management of pulmonary hypertension. Cardiovasc. Res. 44, 274− Cell. Signalling 23, 609−620. 282. (209) Okamoto, M., Murai, J., Yoshikawa, H., and Tsumali, N. (190) Tajsic, T., and Morrell, N. W. (2011) Smooth muscle cell (2006) Bone morphogenetic proteins in bone stimulate osteoclasts hypertrophy, proliferation, migration and apoptosis in pulmonary and osteoblasts during bone development. J. Bone Miner. Res. 21, − hypertension. Comp. Physiol. 1, 295−317. 1022 1033. (191) Lappin, P. B., and Roth, R. A. (1997) Hypertrophy and (210) Steward, C. G., Pellier, I., Mahajan, A., Ashworth, M. T., Stuart, prolonged DNA synthesis in smooth muscle cells characterize A. G., Fasth, A., Lang, D., Fischer, A., Friedrich, W., and Schulz, A. S. pulmonary arterial wall thickening after monocrotaline pyrrole (2004) Severe pulmonary hypertension: a frequent complication of − stem cell transplantation for malignant infantile osteopetrosis. Br. J. administration. Toxicol. Pathol. 25, 372 380. − (192) Lappin, P. B., Ross, K. L., King, L. E., Fraker, P. J., and Roth, R. Hamaetol. 124,63 71. (211) Slatter, M. A., Rao, K., Amrolia, P., Flood, T., Abinun, M., A. (1998) The response of pulmonary vascular endothelial cells to Hambleton, S., Nademi, Z., Goulden, N., Davies, G., Qasim, W., monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro Gaspar, H. B., Cant, A., Gennery, A. R., and Veys, P. (2011) and in vivo. Toxicol. Appl. Pharmacol. 150,37−48. Treosulfan-based conditioning regimens for hematopoietic stem cell (193) Lee, J., Reich, R., Xu, F., and Sehgal, P. B. (2009) Golgi, transplantation in children with primary immunodeficiency (PID): UK trafficking, and mitosis dysfunction in pulmonary arterial endothelial experience. Blood 117, 4367−4375. cells exposed to monocrotaline pyrrole and NO scavenging. Am. J. − (212) Kaplan, F. S., Zasloff, M. A., Kitterman, J. A., Shore, E. M., Physiol.: Lung Cell. Mol. Physiol. 297, L715 L728. Hong, C. X., and Rocke, D. M. (2010) Early mortality and (194) Mukhopadhyay, S., Shah, M., Xu, F., Patel, K., Tuder, R. M., cardiorespiratory failure in patients with fibrodysplasia ossificans and Sehgal, P. B. (2008) Cytoplasmic provenance of STAT3 and PY- progressiva. J. Bone Jt. Surg., Am. Vol. 92, 686−691. STAT3 in the endolysomal compartments in pulmonary arterial (213) Xu, M.-Q., Feldman, G., LeMerrer, M., Shugart, Y. Y., Glaser, endothelial and smooth muscle cells: implications in pulmonary D. L., Urtizberea, J. A., Fardeau, M., Connor, J. M., Triffitt, J., Smith, arterial hypertension. Am. J. Physiol.: Lung Cell Mol. Physiol. 249, R., Shore, E. M., and Kaplan, F. S. (2000) Linkage exclusion and L449−L468. mutational analysis of the noggin gene in patients with fibrodysplasia (195) Bull, L. B. (1955) The histological evidence of liver damage ossificans progressive (FOP). Clin. Genet. 58, 291−298. from pyrrolizidine alkaloids: megalocytosis of the liver cells and (214) Gannon, F. H., Kaplan, F. S., Olmsted, E., Finkel, G. C., inclusion globules. Aust. Vet. J. 31,33−40. Zasloff, M. A., and Shore, E. S. (1997) Bone morphogenetic protein 2/ (196) Jago, M. V. (1969) The development of the hepatic 4 in early fibromatous lesions of fibrodysplasia ossificans progressive. megalocytosis of chronic pyrrolizidine alkaloid poisoning. Am. J. Human Pathol. 28, 339−343. Pathol. 56, 405−422. (215) Enklaar, T., Zabel, B. U., and Prawitt, D. (2006) Beckwith- (197) Svoboda, D., Reddy, J., and Bunyaratvej, S. (1971) Hepatic Wiedemann syndrome: multiple molecular mechanisms. Expert Rev. megalocytosis in chronic lasiocarpine poisoning. Am. J. Pathol. 65, Mol. Med. 8,1−19. 399−408. (216) Sun, J., Liu, Y.-H., Chen, H., Nguyen, M. P., Mishina, Y., (198) Samuel, A., and Jago, M. V. (1975) Localization in the cell Upperman, J. S., Ford, H. R., and Shi, W. (2007) Deficient Alk3- cycle of the antimitotic action of the pyrrolizidine alkaloid lasiocarpine mediated BMP signaling causes prenatal omphalocele-like defect. − and of its metabolite, dehydroheliotridine. Chem.−Biol. Interact. 10, Biochem. Biophys. Res. Commun. 360, 238 243. 185−197. (217) Sotelo-Avila, C., and Gooch, W. M. (1976) Neoplasms (199) Runo, J. R., and Loyd, J. E. (2003) Primary pulmonary associated with the Beckwith-Weidemann syndrome. Perspect. Pediatr. − hypertension. Lancet 361, 1533−1544. Pathol. 3, 255 272. (200) Aldred, M. A., Comhair, S. A., Varella-Garcia, M., Asosingh, K., (218) Rump, P., Zeegers, M. P. A., and van Essen, A. J. (2005) Tumor risk in Beckwith-Wiedemann syndrome: a review and meta- Xu, W., Noon, G. P., Thistlewaite, P. A., Tuder, R. M., Erzurum, S. C., − Geraci, M. W., and Coldren, C. D. (2010) Somatic chromosome analysis. Am. J. Med. Gen. 136A,95 104. abnormalities in the lungs of patients with pulmonary arterial (219) Spector, L. G., and Birch, J. (2012) The epidemiology of hepatoblastoma. Pediatr. Blood Cancer 59, 776−779. hypertension. Am. J. Respir. Crit. Care Med. 182, 1153−1160. (220) Bardeesy, N., Falkoff, D., Petruzzi, M., Nowak, N., Zabel, B., (201) Austin, E. D., Hamid, R., and Ahmed, F. (2010) Somatic Adam, M., Agiar, M. C., Grundy, P., Shows, T., and Pelletier, J. (1994) mutations in pulmonary arterial hypertension. Am. J. Respir. Crit. Care Anaplastic Wilm’s tumour, a subtype displaying poor prognosis, Med. 182, 1095−1095. harbours p53 gene mutations. Nat. Genet. 7,91−97. (202) Taura, P., Garcia-Valdecasas, J. C., Beltran, J., Izquierdo, E., (221) Muwakkit, S., Antillon, F., Valverde, P., Jenkins, J. J., Zaatari, Navasa, M., Sala-Blanch, J., Mas, A., Balust, J., Grande, L., and Visa, J. G., and Dome, J. S. (2002) Letter to the Editor: Simultaneous (1996) Moderate primary pulmonary hypertension in patients − occurrence of Wilms tumor and rhabdomyosarcoma in two patients. undergoing liver transplantation. Anesth. Analg. 83, 675 680. Med. Pediatr. Oncol. 39, 143−145. (203) Hoeper, M. M., Krowka, M. J., and Strassburg, C. P. (2004) (222) Müller-Höcker, J., Weiss, M., Meyer, U., Schramel, P., Portopulmonary hypertension and hepatopulmonary syndrome. Wiebecke, B., Belohradsky, B. H., and Hübner, G. (1987) Fatal − Lancet 363, 1461 1468. copper storage disease of the liver in a German infant resembling (204) Herve, P., Le Pavec, J., Sztrymf, B., Decante, B., Savale, L., and Indian childhood cirrhosis. Virchows Arch. A: Pathol. Anat. Histopathol. Sitbon, O. (2007) Pulmonary vascular abnormalities in cirrhosis. Best 411, 379−385. Pract. Res., Clin. Gastroenterol. 21, 141−159. (223) Price, L. A., Walker, N. I., Clague, A. E., Pullen, I. D., Smits, S. (205) Kochar, R., Moises, I., Rubin, N., and Fallon, M. B. (2011) J., Ong, T.-H., and Patrick, M. (1996) Chronic copper toxicosis Pulmonary complications of cirrhosis. Curr. Gastroenterol. Rep. 13,34− presenting as liver failure in an Australian child. Pathology 28, 316− 39. 320. (206) McDonnell, P. J., Toye, P. A., and Hutchins, G. M. (1983) (224) Seibold-Weiger, K., Vochem, M., Mackensen-Haen, S., and Primary pulmonary hypertension and cirrhosis: are they related? Am. Speer, C. (1997) Fatal hepatic veno-occlusive disease in a newborn Rev. Respir. Dis. 127, 437−441. infant. Am. J. Perinatol. 14, 107−111.

18 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

(225) Sergi, C., Beedgen, B., Linderkamp, O., and Hofmann, W. J. (246) Etzioni, A., Benderly, A., Rosenthal, E., Shehadah, V., (1999) Fatal course of veno-occlusive disease of the liver Auslander, L., Lahat, N., and Pollack, S. (1987) Defective humoral (Endophlebitis Hepatica obliterans) in a preterm infant. Pathol., Res. and cellular immune functions associated with veno-occlusive disease Pract. 195, 847−851. of the liver. J. Pediatr. 110, 549−554. (226) Trollmann, R., Neureiter, D., Lang, T., Dörr, H. G., and (247) Roscioli, T., Cliffe, S. T., Bloch, D. B., Bell, C. G., Mullan, G., Behrens, R. (1999) Late manifestation of Indian childhood cirrhosis in Taylor, P. J., Sarris, M., Wang, J., Donald, J. A., Kirk, E. P., Ziegler, J. B., a 3-year-old German girl. Eur. J. Pediatr. 158, 375−378. Salzer, U., McDonald, G. B., Wong, M., Lindeman, R., and Buckley, M. (227) Bull, L. B., Dick, A. T., Keast, J. C., and Edgar, G. (1956) An F. (2006) Mutations in the gene encoding the PML nuclear body experimental investigation of the hepatotoxic and other effects on protein Sp110 are associated with immunodeficiency and hepatic sheep of consumption of Heliotropium europaeum L.: heliotrope veno-occlusive disease. Nat. Genet. 38, 620−622. poisoning of sheep. Aust. J. Agric. Res. 7, 281−331. (248) Cliffe, S. T., Wong, M., Taylor, P. J., Ruga, E., Wilcken, B., (228) St George-Grambauer, T. D., and Rac, R. (1962) Lindeman, R., Buckley, M. F., and Roscioli, T. (2007) The first Hepatogenous chronic copper poisoning in sheep in South Australia prenatal diagnosis for veno-occlusive disease and immunodeficiency due to the consumption of Echium plantagineum L. (Salvation Jane). syndrome, an autosomal recessive condition associated with mutation − Aust. Vet. J. 38, 288 293. in SP110. Prenatal Diagn. 27, 674−676. (229) Miranda, C. L., Henderson, M. C., and Buhler, D. R. (1981) (249) Cliffe, S. T., Bloch, D. B., Suryani, S., Kamsteeg, E.-J., Avery, D. Dietary copper enhances the hepatotoxicity of Senecio jacobaea in rats. T., Palendira, U., Church, J. A., Wainstein, B. K., Trizzino, A., Lefranc, − Toxicol. Appl. Pharmacol. 60, 418 423. G., Akatcherian, C., Megarbane, A., Gilissen, C., Moshous, D., (230) Howell, J. M., Deol, H. S., Dorling, P. R., and Thomas, J. B. Reichenbach, J., Ziegler, J. B., Wong, M., Tangye, S. G., Lindeman, (1991) Experimental copper and heliotrope intoxication in sheep: R., Buckley, M. F., and Roscioli, T. (2012) Clinical, molecular, and morphological changes. J. Comp. Pathol. 105,49−74. ’ cellular immunological findings in patients with SP110-associated (231) Walker-Smith, J., and Blomfield, J. (1973) Wilson s disease or veno-occlusive disease with immunodeficiency syndrome. J. Allergy chronic copper poisoning? Arch. Dis. Child. 48, 476−479. − ̈ ̈ ̈ Clin. Immunol. 130, 735 742. (232) Muller-Hocker, J., Meyer, U., Wiebecke, B., Hubner, G., Eife, (250) Wang, T., Ong, P., Roscioli, T., Cliffe, S. T., and Church, J. A. R., Kellner, M., and Schramel, P. (1988) Copper storage disease of the (2012) Hepatic veno-occlusive disease with immunodeficiency liver and chronic dietary copper intoxication in two further German (VODI): first reported case in the U.S. and identification of a unique infants mimicking Indian Childhood Cirrhosis. Pathol., Res. Pract. 183, − − mutation in Sp110. Clin. Immunol. 145, 102 107. 39 45. (251) Ganaiem, H., Eisenstein, E. M., Tenenbaum, A., Somech, R., (233) Müller-Höcker, J., Summer, K. H., Schramel, P., and Rodeck, Simanovsky, N., Roscioli, T., Weintraub, M., and Stepensky, P. (2013) B. (1998) Different pathomorphic patterns in exogenic infantile The role of hematopoietic stem cell transplantation in SP110 copper intoxication of the liver. Pathol., Res. Pract. 194, 377−384. associated veno-occlusive disease with immunodeficiency syndrome. (234) Von Mühlendahl, K. E., and Lange, H. (1994) Copper and Pediatr. Allergy Immunol. 24, 250−256. childhood cirrhosis. Lancet 344, 1515−1516. (252) (2009) Genetics Home Reference, US National Library of (235) Scheinberg, I. H., and Sternlieb, I. (1994) Is non-Indian childhood cirrhosis caused by excess dietary copper? Lancet 344, Medicine, http://ghr.nlm.nih.gov/gene/SP110. (253) Deyo, J. A., and Kerkvliet, N. I. (1990) Immunotoxicity of the 1002−1004. pyrrolizidine alkaloid monocrotaline following subchronic adminis- (236) Baker, A., Gormally, S., Saxena, R., Baldwin, D., Drumm, B., − Bonham, J., Potmann, B., and Mowat, A. P. (1995) Copper-associated tration to C57B1/6 mice. Fundam. Appl. Toxicol. 14, 842 849. liver disease in childhood. J. Hepatol. 23, 538−543. (254) Molyneux, R. J., Gardner, D. L., Colegate, S. M., and Edgar, J. ̈ ̈ A. (2011) Pyrrolizidine alkaloid toxicity in livestock: a paradigm for (237) Muller, T., Feichtinger, H., Berger, H., and Muller, W. (1996) − Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder. Lancet human poisoning? Food Addit. Contam., Part A 28, 293 307. 347, 877−880. (255) Newberne, P. M., and Rogers, A. E. (1973) Nutrition, (238) Müller, T., Müller, W., and Feichtinger, H. (1998) Idiopathic monocrotaline and aflatoxin B1 in liver carcinogenesis. Plant Foods − copper toxicosis. Am. J. Clin. Nutr. 67, 1082S−1986S. Man 1,23 31. (239) Wijmenga, C., Müller, T., Brunt, T., Feichtinger, H., (256) Yee, S. B., Kinser, S., Hill, D. A., Barton, C. C., Hotchkis, J. A., Schönitzer, D., Houwen, R. H. J., Müller, W., Sandkuijl, L. A., and Harkema, J. R., Ganey, P. E., and Roth, R. A. (2000) Synergistic Pearson, P. L. (1998) Endemic Tyrolean infantile cirrhosis is not an hepatotoxicity from coexposure to bacterial endotoxin and the allelic variant of Wilson’s disease. Eur. J. Hum. Genet. 6, 624−628. pyrrolizidine alkaloid monocrotaline. Toxicol. Appl. Pharmacol. 166, − (240) Tanner, M. S. (1998) Role of copper in Indian childhood 173 185. cirrhosis. Am. J. Clin. Nutr. 67, 1074S−1081S. (257) (1992) Abwehr von Arzneimittelrisiken-Stufe II. Bundesan- − (241) Aston, N. S., Morris, P. A., Tanner, M. S., and Variend, S. zeiger 17 June, 4805; cited by Dtsch. Apoth. Ztg. 132, 1406 1408. (1998) An animal model for copper-associated cirrhosis in infancy. J. (258) European Medicines Agency (2013) Public statement on the use Pathol. 186, 215−221. of herbal medicinal products containing toxic, unsaturated pyrrolizidine (242) Dieter, H. H., Schimmelpfennig, W., Meyer, E., and Tabert, M. alkaloids (PAs), EMA/HMPC/893108/2011, 2nd draft, 6 Nov, (1999) Early childhood cirrhosis (ECC) in Germany between 1982 EMA,London, http://www.ema.europa.eu/docs/en_GB/document_ and 1994 with special consideration of copper etiology. Eur. J. Med. library/Public_statement/2013/11/WC500154224.pdf. Res. 28, 233−242. (259) Czauderna, P., Katski, K., Kowalczyk, J., Kurylak, A., Lopatka, (243) Zietz, B. P., de Vergara, J. D., and Dunkelberg, H. (2003) B., Skotnicka-Klonowicz, G., Sawicz-Birkoska, K., and Godzinski, J. Copper concentrations in tap water and possible effects on infant’s (2000) Venoocclusive liver disease (VOD) as a complication of healthresults of a study in Lower Saxony, Germany. Environ. Res. 92, Wilms’ tumour management in the series of consecutive 206 patients. 129−138. Eur. J. Pediatr. Surg. 10, 300−303. (244) Zietz, B. P., Dieter, H. H., Lakomek, M., Schneider, H., (260) Cecen, E., Uysal, K. M., Ozguven, A., and Gunes, D. (2007) Kessler-Gaedke, B., and Dunkelberg, H. (2003) Epidemiological Veno-occlusive disease in a child with rhabdomyosarcoma after investigation on chronic copper toxicity to children exposed via the conventional chemotherapy. Pediatr. Hematol. Oncol. 24, 615−621. public drinking water supply. Sci. Total Environ. 302, 127−144. (261) Xia, Q., Zhao, Y., Von Tungeln, L. S., Doerge, D. R., Lin, G., (245) Mellis, C., and Bales, P. M. (1976) Familial hepatic Cai, L., and Fu, P. P. (2013) Pyrrolizidine alkaloid-derived DNA venoocclusive disease with probable immune deficiency. J. Pediatr. adducts as a common biological biomarker of pyrrolizidine alkaloid- 88, 236−242. induced tumorigenicity. Chem. Res. Toxicol. 26, 1384−1396.

19 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20 Chemical Research in Toxicology Perspective

(262) Gao, H., Li, N., Wang, J. Y., Zhang, S. C., and Lin, G. (2012) Definitive diagnosis of hepatic obstruction syndrome induced by pyrrolizidine alkaloids. J. Dig. Dis. 13,33−39.

20 DOI: 10.1021/tx500403t Chem. Res. Toxicol. 2015, 28, 4−20