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Perspective

Pyrrolizidine Alkaloids: Potential Role in the Etiology of Cancers, Pulmonary Hypertension, Congenital Anomalies and Liver Disease. John Alexander Edgar, Russell John Molyneux, and Steven Mark Colegate Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500403t • Publication Date (Web): 06 Dec 2014 Downloaded from http://pubs.acs.org on December 11, 2014

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Chemical Research in Toxicology

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 Ave, North Ryde, NSW 2113, Australia. ‡College of

Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720 United States. §

Poisonous Plants Research Laboratory, ARS/USDA, 1150 East 1400 North, Logan, UT

84341 United States.

KEYWORDS: 1,2-dehydropyrrolizidine; food contamination; dietary exposure; chronic disease; sinusoidal obstruction; genotoxicity; teratogenicity; rhabdomyosarcoma; cirrhosis; bone morphogenetic protein signaling.

*Corresponding Author. Tel: +61 2 9490 5000; Fax: +61 2 9490 8499; E-mail: [email protected]

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ACS Paragon Plus Environment

OH O

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Hepatotoxic Pneumotoxic Genotoxic Teratogenic

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milk, eggs, Liver Disease breads, teas Cancers Pulmonary Hypertension Congenital Anomalies

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ABSTRACT: Large outbreaks of acute food-related poisoning, characterized by hepatic sinusoidal obstruction syndrome, hemorrhagic 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 1,2dehydropyrrolizidine 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 1,2dehydropyrrolizidine 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; 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 1,2dehydropyrrolizidine alkaloids is a significant cause of some of these costly and debilitating diseases, this should lead to initiatives to reduce the level of these alkaloids in the food chain. 3



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1. INTRODUCTION 1,2-Dehydropyrrolizidine ester alkaloids (dehydroPAs) have been known for almost a century to be the cause of large and recurring episodes of acute hepatotoxicity in a number of countries.1-16 The alkaloids responsible for these outbreaks of poisoning are produced by weeds that often infest grain crops and the primary cause is consumption of bread made using dehydroPA-contaminated flour. Typical infestations of dehydroPA plants are shown in Figure 1 Episodes of acute dehydroPA toxicity involving the consumption of bread are avoided in many countries by more effective control of weeds in crops and by strictly applying food safety standards limiting the number of foreign seeds in grain entering the human food chain, including some known to contain dehydroPAs. Flour millers also normally screen grain prior to milling to ensure that all foreign seeds and plant fragments are removed. However, if dehydroPA-containing plants were present in the crop, dust containing dehydroPAs is not removed by screening and some dehydroPAs remain associated with the grain.17-20 It is normal practice in countries undertaking large scale cereal production to combine grain from many farms and from different cropping areas. Such bulking dilutes and reduces the level of unwanted contaminants in the grain, including dehydroPAs, but it results in a wider distribution of any dehydroPAs that were present and thus increases the population exposed to low levels of these hazardous chemicals in grain-based foods. While these measures and practices are apparently sufficient to prevent the acute dehydroPA poisoning that regularly occurs in countries in which such measures and practices are not applied, it is still possible that occasional, low level dietary exposure in grain-based products could be contributing worldwide to the incidence of several slowly developing, chronic diseases.18,19 Other food items increasingly being reported to be periodically contaminated by low levels of dehydroPAs include milk, eggs, meat, honey, pollen, teas, salads and foods 4


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containing these as ingredients.18,20-40 Both Echium spp. shown in Figure 1, and a number of other dehydroPA producing plants, are widely used in commercial honey production41 and the honey produced is significantly contaminated by dehydroPAs, e.g. up to 2000 - 4000µg of dehydroPAs/kg in some supermarket honeys.28,29,40 Levels of dehydroPAs up to 5647 µg/kg have recently been reported in teas purchased in retail markets in Germany.21 The levels of dehydroPAs found in these foods are generally too low to cause acute hepatotoxicity.18,31,32,4244

However, based on a consideration of their well-established genotoxicity,8,45,46 and on

recent risk assessments suggesting that to make cancer unlikely a maximum tolerable daily intake of 0.007µg of dehydroPAs/kg body weight (bw) should not be exceeded,31,42-44 the dehydroPAs in these foods could be contributing to somatic mutations leading to a wide range of cancers.8,46 They may also be initiating other chronic diseases such as pulmonary arterial hypertension (PAH) leading to enlargement of the right ventricle and right heart congestive failure8,20,47 and be a cause of some congenital anomalies.8,20,47-49 This Perspective provides a comprehensive review of the characteristics of acute and chronic dehydroPA toxicity and suggests some associative considerations, or indicators, for linking specific cases of cancer, PAH, congenital anomalies and liver diseases with intermittent, low level dietary exposure to dehydroPAs. Some of these indicators are then used to identify cases of chronic diseases reported in the literature that may have resulted from dietary exposure to dehydroPAs. The etiology of these cases warrants further investigation to confirm or negate this possibility. Many cases of cancer, PAH, congenital anomalies and liver disease are of unknown etiology so that establishing they are sometimes caused by dietary exposure to dehydroPAs should lead to ways of reducing the incidence of these costly and debilitating medical conditions. The challenge is to identify specific cases that have been initiated or exacerbated by dietary exposure to dehydroPAs. 5



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2. TOXICOLOGY 2.1 Occurrence, Evolution and Chemistry. It has been estimated that 3% of all flowering plants produce poisonous dehydroPAs.50 They are very effective insect-feeding deterrents and consequently have evolved independently on at least four occasions in a number of different plant families.51 Approximately 500 potentially toxic dehydroPAs are known.4,52 Varying in the character and/or stereochemistry of the necine base (Figure 2) and the esterifying acids (necic acids), they can be classified into four main categories (Figure 2): a) monoesters (e.g. heliotrine); b) open chain diesters (e.g. lasiocarpine); c) macrocyclic diesters (e.g. retrorsine); and d) seco-alkaloids (e.g. senkirkine). This complexity is further amplified by their existence in plants as both free bases and as N-oxides (Figure 2), the latter predominating in many plant species.4,52 2.2 Bioactivation and Mechanism of Toxicity. DehydroPA free bases are protoxins. Following ingestion, they are absorbed from the gut and converted in the liver, by cytochrome P450 monooxygenases (CYP450), specifically the CYP3A and CYP2B isoforms in humans, to 1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine ester metabolites (DHP esters) (Figure 3) 8,46,52-75. These “pyrrolic” metabolites are bifunctional biological alkylating agents capable of causing tissue damage and inducing genetic mutations.52 In vivo Noxidation of dehydroPAs by CYP450 enzymes and flavin-containing mono-oxygenases68 is a detoxifying mechanism leading to excretion of the water soluble dehydroPAs N-oxides produced (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

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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 on other substances, e.g. glutathione (GSH), in the liver to produce mixtures of C7 and C9 mono and di DHP adducts, the latter forming crosslinks between –SH, –OH and –NH groups on different molecules or on the same macromolecule (Figures 3). 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 dehydroretronecine,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 metabolism.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.4,25,52,90,98,99,100-103 DHP, the final and common toxic metabolite of all dehydroPAs, is carcinogenic,104-106 immunosuppressive107 and mimics the anti-mitotic effects of radiation in tissues displaying high rates of mitosis.108-111 Circulating DHP is considered to be responsible for the effects of 7



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dehydroPAs outside of the liver. It is however highly water soluble and any DHP that remains un-adducted is excreted (Figure 3).52 The potential harm of dietary exposure to dehydroPAs does not end with formation of the initial DHP adducts or excretion of dehydroPA N-oxides and DHP. Mono DHP adducts, retaining one active alkylating site at C7 or C9, remain potentially hazardous as alkylating agents in vivo.112 In accord with this, a mixture of preformed galectin-1-DHP adducts (Figure 4), shown by mass spectrometry to be mainly mono-adducted, was found to be cytotoxic in human pulmonary artery endothelial cells.112 More importantly, it has been established that some DHP adducts, involving DHP attached to weak nucleophiles, can dissociate and release DHP or transfer DHP to stronger nucleophilic sites.52,95-97,109 The in vivo persistence of a “reservoir of stabilized pyrrolic alkylating agents (DHP adducts) which could subsequently interact with other tissue constituents”52 is one reason why the toxic effects of dehydroPAs continue to develop slowly over an extended period even from a single exposure to these hazardous substances.52,95-97,109-111 The reversibility of the reaction of dehydroPA metabolites with certain weak nucleophiles prolongs the effects and enhances the hazard of dietary exposure to dehydroPAs relative to some other genotoxic substances and this fact should be taken into account in assessing the risk of foods contaminated by these alkaloids.

3. PATHOLOGICAL EFFECTS 3.1. Sinusoidal Obstruction Syndrome and Cirrhosis. Following exposure to relatively high doses of dehydroPAs, e.g., tens of milligrams of dehydroPAs/ kg bw, the liver sinusoidal endothelial cells have been shown, in animal models, to be the first to display the effects of the DHP ester metabolites produced in adjacent hepatocytes. They have been observed to round-up, swell and slough off, leading to blockage of the microcirculation in the sinusoids.114-118 The resulting hepatic sinusoidal obstruction syndrome (HSOS) (also referred 8


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to as hepatic veno-occlusive disease or VOD) is highly characteristic of acute dehydroPA poisoning seen in people8,14 and leads to necrosis of surrounding liver tissue, fibrosis, nodular hyperplasia, bile duct proliferation and eventually to cirrhosis and liver failure.8,110,117-119 DehydroPAs are the only naturally occurring agents in the environment known to produce HSOS when consumed and they are frequently used to produce animal models of clinical cases of HSOS and chronic, progressive liver disease that are used to study the development of, and potential treatments for, these diseases.115,118,120,121 The particular susceptibility of the sinusoidal endothelial cells is thought to be, in part, associated with increased activity of matrix metalloproteinases (MMPs), e.g. MMP-9 (gelatinase B), that degrade the extracellular matrix leading to the endothelial cells being released.116,117,122 MMPs are normally suppressed by GSH. As sinusoidal GSH levels rapidly decrease due to alkylation by dehydroPA metabolites and the redox state of the sinusoids changes, the activity of the MMPs increases and HSOS develops. In support of this mechanism, it has been demonstrated, for example, that GSH infused into the portal vein prevents HSOS from developing in animals exposed to dehydroPAs.116,123 While HSOS is considered to be highly indicative of dehydroPA exposure8,111,119 and has developed in humans consuming levels as low as tens of micrograms of dehydroPAs/kg bw/day,17,110,124,125 it is not always readily apparent. For example, when the liver damage develops over an extended period, due to relatively low level exposure to dehydroPAs, HSOS may not be apparent or may be obscured by hepatic fibrosis and nodular regeneration.110 Under these circumstances, the resulting cirrhosis may be indistinguishable from cirrhosis caused by other agents, e.g. alcohol, aflatoxins and hepatitis viruses, and the etiology can be ambiguous.110,119 Thus, as well as clinical cases of HSOS, some cases of cirrhosis of unknown cause and without obvious HSOS, could also have a dietary dehydroPA etiology.

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3.2 Pulmonary Arterial Hypertension (PAH). Recognition that ingestion of dehydroPAs causes PAH leading to enlargement of the right ventricle and right heart failure (cor pulmonale) was established many years ago in experimental animals56,126-133 and since then dehydroPAs such as monocrotaline and fulvine have been deliberately administered by medical researchers to various animal species to generate experimental models of PAH.134-139 Doses of dehydroPAs required to produce PAH in experimental animals comparable to clinical cases of PAH in humans are generally lower than those required to produce HSOS. This may explain therefore the occasional development of PAH, without notable liver pathology, in people taking herbal medicines containing dehydroPAs.140-142 Children form a significant cohort of PAH cases of unknown etiology. The PAH produced in non-human primates by monocrotaline shows very similar pathology to PAH diagnosed in children.130-132 Rodents given monocrotaline are apparently a less appropriate model of the human disease131,132,136 but the monocrotaline-rodent model has become the most widely used to study the development of PAH and to test treatments for this condition.137,138 Stenmark et al.136 and Gomez-Arroyo et al.139 have discussed some of the differences between the monocrotaline-rodent models of PAH and PAH seen in humans. These differences appear to relate to the initial acute effects of dehydroPAs that are not seen in clinical cases of PAH. These discordant effects could be explained, in part, by the rodent model of PAH typically being generated using a single, relatively high dose of monocrotaline, sufficient to produce PAH relatively quickly in a high proportion of animals and not just the most susceptible. A long term, intermittent, lower level of exposure, similar to that expected from the levels of dehydroPAs currently being detected in food, may avoid the early discordant acute effects sometimes seen in the standard monocrotaline-rodent model of PAH136,139 and may be expected to slowly produce PAH more typical of the disease

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normally seen in humans and in the non-human primate model developed by Chesney and Allen.130-132 Several experiments with non-human primates provide insights into what can be expected from long term, low level dietary exposure to dehydroPAs.130-132 A highly significant, age-related difference was observed following subcutaneous injection of the relatively high dose of 30 mg/kg bw monocrotaline, followed by three further injections, 2 months apart, of 60 mg/kg bw, to 4-week old and 15-month old monkeys.130 The older animals, as expected from the very high doses administered, developed HSOS and showed typical dehydroPA liver pathology while the young monkeys showed minimal liver pathology but developed PAH typical of that seen in children. It was established that the young monkeys displayed much lower cytochrome CYP450 activity in their livers and would therefore have produced insufficient levels of the monocrotaline DHP ester metabolite (dehydromonocrotaline) to rapidly deplete sinusoidal GSH, activate MMPs, release sinusoidal endothelial cells and cause HSOS. They must however have produced sufficient low levels of circulating, labile DHP adducts and DHP to damage the endothelium of the pulmonary arteries and cause PAH. The response of the younger monkeys represents the situation that would most likely pertain if a human diet regularly delivered very small amounts of dehydroPAs that yielded insufficient toxic metabolites to cause HSOS but sufficient to initiate PAH and perhaps cause somatic mutations leading to other genotoxic conditions such as cancer. Thus PAH and cancer, rather than HSOS, are the most likely outcomes from circulating low µg/kg bw levels of labile DHP adducts and free DHP. This would be especially so if dietary exposure to dehydroPAs is intermittent since this would allow the well established antimitotic effects of dehydroPA metabolites to be relatively shortlived and enable cancers and progressive PAH, both requiring cell proliferation, to develop. This type of intermittent, low level dietary exposure seems increasingly likely to have 11



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occurred in the past and to continue to the present day to varying degrees in many regions throughout the world.20,47 Current knowledge of the relatively low but significant levels of dehydroPAs in food and an expectation of intermittent dietary exposure, insufficient to produce HSOS, suggests that dietary dehydroPAs could be responsible for at least some cases of clinical PAH of currently unknown etiology reported in the medical literature. 3.3 Carcinogenesis. It has been shown, using experimental animals and human cell cultures, that one of several genetic targets for toxic dehydroPA metabolites is the gene TP53 encoding the tumor suppressor protein p53.46,143,144 Signature mutations of dehydroPAs include G:C→T:A transversion and tandem base substitutions.46 Cancers of the liver, lung, kidney, skin, intestines, bladder, brain and spinal cord, pancreas, adrenal gland, muscle (rhabdomyosarcoma, RMS) and blood (leukemia) have been produced by dehydroPAs and their metabolites in experimental animals.8,46,145,146 These are also the tissues in which DHP adducts have been detected. A very similar, wide range of cancers is associated with LiFraumeni Syndrome caused by germline mutations of TP53147 a known target of dehydroPA metabolites. As previously mentioned, dehydroPAs and their DHP metabolites display strong antimitotic effects8,52,106 typical of many other biological alkylating agents used to treat cancer. A dehydroPA-N-oxide, indicine-N-oxide, was at one time clinically investigated for use as a cancer chemotherapeutic agent.148,149 The anti-mitotic effect of dehydroPAs exposure can therefore be expected to initially inhibit both cancer development and the development of the “quasi-malignant” endothelial cells in the pulmonary arterioles that characterize PAH.150 The development of both cancer and PAH will therefore be particularly favored by extended, low level and intermittent periods of dehydroPA exposure, as is expected from current knowledge of foods contaminated by dehydroPAs, since this type of exposure allows intervening periods

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of normal mitotic activity that provide opportunity for cancers and progressive PAH to develop.8,52,110,150-155 3.4 Teratogenicity. Exposure of pregnant women to dehydroPAs in herbal teas, herbal medicines and spices has caused fatal HSOS in neonates.8,52,110,125 DehydroPAs and their DHP metabolites are teratogenic in rats and it has been shown that they cross the placenta and form DHP adducts in rat embryos.48,49,52 Growth retardation, abnormal skeletal development and deficiencies in ossification are common features. Congenital anomalies have however not yet been extensively studied or considered as a possible consequence of low level dietary exposure to dehydroPAs. Nonetheless, as well as stillbirths and HSOS, congenital anomalies are to be expected in surviving neonates following dietary exposure of their mothers to dehydroPAs during pregnancy.

4. POSSIBLE INDICATORS OF DEHYDROPYRROLIZIDINE ALKALOID INVOLVEMENT Growing evidence of significant dehydroPA contamination of a wide range of common foods suggests that they could be a common cause of several chronic diseases.20 The ability of dehydroPAs to cause disparate chronic diseases, viz. cancers, PAH, HSOS and genetic anomalies, suggests that disease complexes involving these diseases could be used to indicate the involvement of dehydroPAs. This section considers the identification of diseases potentially caused by dietary dehydroPAs in this context. 4.1 Cancer. Linking a diverse range of cancers with somatic mutations arising from relatively constant or, more effectively, intermittent, low-level, long term dietary exposure to dehydroPAs is likely to be difficult but the highly characteristic ability of dehydroPAs to produce HSOS, PAH and genetic anomalies could be used as clinical signs suggesting such a

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link. Evidence of one or more of these other conditions can be expected in at least some patients with cancers caused by dehydroPAs. As well as exposure to dehydroPAs, another clinically recognized and common cause of dehydroPA-like HSOS is associated with chemotherapy of childhood cancers such as rhabdomyosarcoma (RMS), nephroblastoma (Wilms tumor), metastatic colorectal cancer and leukemia using alkylating agents such as cyclophosphamide or busulfan.156-164 Treatment of hematologic cancers in neonates by allogenic stem cell transplantation (SCT), following myeloablation using these agents can also cause HSOS as a complication.164-177 Like dehydroPAs, cyclophosphamide and busulfan are biological alkylating agents and thus may simply mimic dehydroPAs in causing HSOS178-180 although, unlike dehydroPAs, neither of these agents is used to produce animal models of HSOS. Indeed, cyclophosphamide is not generally considered to be hepatotoxic 181 so it is somewhat unexpected that it causes HSOS in some childhood cancer patients. It is however possible that if the cancer being treated developed as a consequence of dietary exposure to dehydroPAs, for example when fetuses are exposed to dehydroPA metabolites in utero or as infants consuming dehydroPA-contaminated breast milk and/or dehydroPA-contaminated food, asymptomatic HSOS may co-occur with their cancers. Overt HSOS can then be precipitated by the additional insult to the sinusoids from chemotherapeutic agents not normally capable of causing HSOS. Dietary exposure to low levels of dehydroPAs may be, like radiotherapy, a priming event for HSOS when cancers caused by dehydroPAs are treated with therapeutic alkylating agents such as cyclophosphamide and busulfan. DehydroPAs have been shown to induce biosynthesis of GSH in the liver in response to the depletion of GSH they cause.182 The potential genotoxicity associated with continued dietary exposure to dehydroPAs during induction of GSH biosynthesis could cause mutations in the upregulated GSH biosynthesis genes. In turn, this could lead to compromise of the 14


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ability of some dehydroPA-affected livers to biosynthesize GSH and thus dehydroPAexposed livers could become sensitized to other alkylating agents such as cyclophosphamide or busulfan that normally do not cause HSOS. In this regard it would be of interest to compare the development of HSOS in experimental animals using cyclophosphamide and busulfan with and without priming by dietary exposure to very low levels of dehydroPAs, and also to investigate whether the GSH biosynthesis capability of livers is compromised by such priming. Rhabdomyosarcoma (RMS) is a cancer originating from mutations in muscle progenitor cells.183 DHP becomes a more aggressive alkylating agent under mildly acid conditions due to protonation of the alcohol groups providing a good leaving entity (H2O) for generating the carbonium ions involved in alkylation.52,87,95 Therefore accumulation of lactic acid in muscle tissue could quite conceivably result in myoblasts being a favored site for DHP alkylation causing mutations and resulting in RMS. In accord with this possibility, while intra-peritoneal injection of DHP into rats produces a range of cancers similar to those produced by ingestion of the parent dehydroPAs,106 subcutaneous injection of the DHP enantiomer dehydroretronecine produced RMS at the site of injection in 65% of rats (39 of 60).104,105 A dehydroPA etiology for some cases of RMS is therefore in accord with this observation and also with HSOS being a complication in a significant proportion of RMS patients when they undergo chemotherapy involving alkylating agents such as cyclophosphamide and busulfan. Cancer clusters involving several types of cancer could also be indicative of a dietary dehydroPA origin. Such heterogeneous clusters are often dismissed as unlikely to have a common etiology. However, Daughton184 has drawn attention to the co-occurrence of acute lymphoblastic leukemia and RMS in Nevada and Arizona and has suggested that this coincidence could indicate a dietary dehydroPA etiology. It would be of interest to determine 15



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if there was concurrently or subsequently an increased incidence of cirrhosis, HSOS and PAH in those communities. 4.2 Pulmonary Arterial Hypertension. Dysregulation of bone morphogenetic protein (BMP) signaling is a feature of clinical cases of both sporadic (idiopathic) and inherited (familial) PAH and has been implicated in the vascular cell proliferation and remodeling of pulmonary arterioles seen in PAH.185 The dehydroPA monocrotaline and its DHP ester metabolite (dehydromonocrotaline, “monocrotaline pyrrole”, Figure 4) cause mutation of BMP signaling genes in animal models of PAH185-187 and their ability to cause mutations of BMP-signaling genes is clearly a significant factor in the ability of dehydroPAs to cause PAH. A hallmark feature of both clinical PAH and dehydroPA-induced PAH are the proliferative, “quasi malignant”, hypertrophic (megalocytic), apoptosis-resistant cells, in particular smooth muscle cells, that contribute to the characteristic plexiform (onion skinlike) lesions responsible for progressive vascular remodeling and vasoconstriction of the pulmonary arterioles.138,188-194 The previously mentioned susceptibility of muscle cells to DHP alkylation leading to RMS may have a parallel in the development of the quasimalignant smooth muscle cells in the pathogenesis of PAH. In regard to the hypertrophic character of the pulmonary artery endothelial cells involved in PAH, similar long-lived, morphologically and functionally viable megalocytes have long-been recognized as a highly characteristic aberration of hepatocytes, in dehydroPA-poisoned animals.4,52,195-198 They have also been described in the lungs,152 kidney4,152 and duodenum111 of animals exposed to dehydroPAs. While functionally viable and long-lived, the hepatic megalocytes that develop in surviving animals following acute dehydroPA-poisoning, initially appear incapable of division.196,197 This characteristic is however believed to be due to the persistent anti-mitotic effects of dehydroPAs and, given 16


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time, some of these hypertrophic cells are expected to “escape from mitotic inhibition”.197 In some cases the proliferative, hypertrophic, apoptosis-resistant smooth muscle cells, seen in the pulmonary arterioles of patients with PAH could arise in this way and be indicative of intermittent, low level dietary exposure to dehydroPAs. Twenty percent of idiopathic PAH cases are associated with BMP receptor II (BMPR2) mutations and 75% of familial PAH cases display germline mutations of BMPR2.199,200 These mutations alone are however insufficient to cause PAH.199-201 The low penetrance (20%) of PAH among carriers of BMPR2 mutations has led to the suggestion that additional environmental factors and further somatic mutations may be needed before PAH develops in people carrying BMPR2 mutations. It has therefore been suggested that a socalled “second hit” is required before PAH develops.200,201 Dietary exposure to dehydroPAs could deliver such a “second hit” in carriers of BMPR2 mutations and lead to the development of PAH in these individuals. Thus dietary exposure to dehydroPAs may not only be responsible for the development of PAH in previously mutation-negative individuals it could also be an important contributor to the development of PAH in carriers of BMPR2 mutations. Portopulmonary hypertension (POPH) is a relatively rare form of PAH associated with cirrhosis and portal hypertension.202-204 The exact pathogenesis and etiology of POPH is poorly understood.205 One clinical study of 2,459 patients with cirrhosis showed that15 (0.61%) exhibited POPH and concluded that an unknown dietary cause was “the most attractive explanation for the association of portal and pulmonary hypertension”.206 Another study207 has suggested that the “prevalence of primary pulmonary hypertension associated with cirrhosis may be greater than previously reported.” The combination of cirrhosis, portal hypertension and PAH, characteristic of POPH, resembles the chronic condition sometimes

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caused by dehydroPAs in animals, so that dietary dehydroPAs could conceivably be, in some cases, a differential consideration in determining the etiology of POPH-like diseases. All cases of PAH of uncertain etiology need to be investigated as being possibly caused, initiated or exacerbated by dietary exposure to dehydroPAs. 4.3 Congenital Anomalies. The skeletal defects seen in rat fetuses whose mothers were exposed to heliotrine or DHP48,49 strongly suggests that the alkaloids are causing deficiencies in BMP signaling.208, 209 As previously discussed, mutation of BMP signaling genes by dehydroPA metabolites contributes to their ability to cause PAH in experimental animals, so that it is not surprising that ineffective bone formation is amongst the congenital anomalies seen in animals exposed to dehydroPA metabolites in utero.48,49 HSOS is commonly experienced as a complication of conditioning regimes involving cyclophosphamide and busulfan used for prior marrow ablation when, for example, congenital anomalies such as malignant infantile osteopetrosis (MIOP), primary immunodeficiency (PID) and severe immune dysregulatory disorders (SIDs) are treated by SCT.210,211 If these congenital anomalies developed as a consequence of maternal exposure to dehydroPAs, such exposure could prime the livers of the neonates for development of HSOS during myeloablation. As well as displaying a predisposition to developing HSOS following SCT, some MIOP patients also frequently develop severe PAH as a complication.210 PAH was also the most common cause of death in a study of 48 patients with fibrodysplasia ossification progressiva (FOP).212 Of the 48 FOP patients studied, 26 (54%) died prematurely (age range 8-58, median life span 42 yrs) from PAH. Another possible indication of a dehydroPA etiology is that FOP is characterized by congenital toe malformations and ossification issues involving BMP signaling abnormalities.213,214

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Beckwith-Wiedemann Syndrome (BWS), some features of which involve faulty BMP signaling,215,216 is another congenital disorder that could sometimes have a dehydroPA etiology. BWS is associated with significantly increased risk of tumors such as RMS, hepatoblastoma and Wilms tumor,217-219 including, in some cases, the co-occurrence of RMS and the anaplastic subtype of Wilms tumor associated with mutations of the TP53 gene.220,221 The deficiencies in BMP signaling, the susceptibility to developing certain cancers, including RMS, and concurrent susceptibility to HSOS during chemotherapy could quite conceivably indicate that the congenital defects of BWS, the cancers that develop and the susceptibility to HSOS may sometimes all have a common dehydroPA origin. In combination, these observations suggest that a dehydroPA etiology should be considered as a possible cause of some cases of BWS, MIOP, FOP, PID and SID and possibly other congenital conditions. 4.4 Liver Disease. Dietary exposure to dehydroPAs should also be considered for all cases of cirrhosis of unknown etiology, and especially those showing evidence of HSOS. While clinicians dealing with cases of HSOS, especially in neonates and young children, often questioned the mothers of the patients involved in regard to their consumption of herbal medicines containing dehydroPAs,222-226 the potential for dehydroPAs being present in foods they have consumed, such as milk, honey, flour, tea and eggs, is not normally considered. Environmental copper is known to exacerbate the hepatotoxicity of dehydroPAs in livestock and to result in accumulation of very high levels of copper in the cirrhotic liver.4,52,77,227-230 The poisoning of livestock by dehydroPAs was at one time called “chronic copper toxicity” until it was shown that this particular condition also required exposure to plants containing dehydroPAs.4,227 Numerous investigations have sought to explain individual examples and clusters of cirrhosis in young children that have occurred in Germany, Austria, India, the USA, Australia and elsewhere. Such cases are sometimes associated with copper 19



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accumulation.222-226, 231-242 Many investigations have concluded however that high levels of environmental copper are not the only cause.235,237,238 Some have suggested that other environmental agents, including exposure to dehydroPAs,240,241 and perhaps a non-Wilsonian genetic predisposition to copper accumulation and toxicity, may be required.234,236-240,242-244 This situation is very reminiscent of the historic debate in the 1950s on the etiology of “chronic copper toxicity” in livestock.4,52,227 Cirrhosis accompanied by copper accumulation is therefore another important indication for suspecting possible dietary dehydroPA involvement. A form of HSOS called “hepatic veno-occlusive disease with immunodeficiency” (VODI) is associated with germline mutations of the SP110 nuclear body protein gene.245-250 SP110 nuclear body protein is involved in regulation of the body’s immune response so that a non-functional version of SP110 nuclear body protein will lead to impairment of the immune system.251 It is however less clear how the loss of SP110 nuclear body protein can cause HSOS.252 This suggests a possible role for another factor such as dietary dehydroPAs. The known immunotoxicity of dehydroPAs and DHP107,108,253 could conceivably contribute to the development of a VODI-like disease in susceptible individuals with or without a pre-existing germline mutation of the SP110 gene. It is even possible that mutation of the SP110 gene by dehydroPA metabolites could contribute to or account for some of the immune system impairment associated with dehydroPA exposure.107 4.5 Exacerbating Factors. Modern bulking of commodities such as grain, milk and honey for wide national and international distribution leads to dilution of dehydroPA contaminants and ensures that dietary exposure to hazardous levels of dehydroPAs is relatively rare in the many countries where such bulking normally occurs. Thus hazardous levels of dietary exposure to dehydroPAs are likely to occur more commonly in particular localities and communities or households due to specific items of food produced and 20


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consumed locally being contaminated by dehydroPAs at certain times. Regional and/or familial clusters of dehydroPA-associated chronic diseases are particularly likely in areas where locally produced and consumed honey, milk, eggs, grain and other foods are occasionally contaminated via dehydroPA-producing plants that grow in that region and especially during times when incidents of livestock poisoning are being reported.254 A familial genetic propensity for hepatic activation of dehydroPAs, rather than detoxication, associated with inherited CYP450 profiles can also be expected to result in family clusters of dehydroPA-initiated chronic diseases. Some medications, e.g. rifampicin, St Johns wort and phenobarbital and environmental contaminants, e.g. polychlorinated biphenyls, induce cytochrome CYP3A enzymes, so that concurrent consumption or exposure to these could increase an individual’s susceptibility to dehydroPA toxicity via enhanced bioactivation. Viruses, bacterial endotoxins and aflatoxins have also been shown to exacerbate liver damage and cancers caused by dehydroPAs.255,256 The particular susceptibility of fetuses and nursing neonates to toxic effects when their mothers consume foods or herbal medicines containing dehydroPAs is well established.8, 20, 257,258 Fetal and neonate liver CYP450 enzymes are generally less effective in activating dehydroPA52,130 and it is thought that the DHP metabolites produced by and transferred from the mother are largely responsible for the harm caused to fetuses and breast feeding neonates.52 In recognition of the susceptibility of fetuses and nursing neonates, German regulations for phytopharmaceuticals specify that dehydroPA containing herbal medicines cannot be sold to pregnant or breast feeding women.257,258 For other consumers in Germany the daily dose of dehydroPAs in a small number of specified dehydroPA-containing phytopharmaceuticals must not exceed 1µg, reduced to 0.1µg if the medication is taken daily for more than six weeks per year.257 These regulations are not currently applied to food 21



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although a significant number of retail food items have been shown to exceed the maximum levels of dehydroPAs allowed in phytopharmaceuticals in Germany.20,27-29,33-40

5. SELECTED INDICATORS SUGGESTING A POSSIBLE DEHYDROPYRROLIZIDINE ALKALOID ETIOLOGY It will be necessary to eventually develop criteria, well supported by targeted research and epidemiological studies, which can be used to assist in identifying diseases which may be associated with dietary exposure to dehydroPAs. Until this is achieved, the following indicators, rationally based on associative considerations described above, are suggested as possible indications of a dietary dehydroPA etiology and to suggest the need for further investigations into possible sources of exposure: 1) Latent or overt HSOS and PAH of unknown or uncertain etiology. 2) Cirrhosis, especially if associated with HSOS and/or accumulation of copper in the liver. 3) Cancers and/or congenital anomalies where there is evidence of overt or asymptomatic HSOS, PAH, bone deformities or immunological deficiencies. 4) PAH accompanied by evidence of hepatotoxicity. 5) Any of the above occurring in consanguineous or regional clusters, especially in areas where dehydroPA-producing weeds grow and where locally produced dehydroPA-contaminated foods are likely to occasionally occur and especially if there is concurrent evidence of livestock being poisoned by dehydroPAs.254 6) Any of the above involving fetuses, neonates and children.

In considering and selecting these indicators, several cases of liver disease and cancers with a possible dehydroPA etiology have been identified.158-160,163,168,176,222-226,23122


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234,236-238,245,247-249,259,260

Of these, ten cases159,160,176,223,237,238,245,247-249 display at least four of

the suggested indicators, nine others158,163,222,224,225,232,233,258,260 have three indicators and five other cases168,226,231,234,236 have two of the suggested indicators. Some of these cases also showed other features, including hypertrophic hepatocytes ("ballooned", "swelling"),222,232,233,237,238 inconsistent "veno-occlusive disease" (HSOS)238 and "rare mitotic figures",223 that are indicative of a dehydroPA etiology. If several of the indicators suggested above are present in an individual case the possibility that dietary exposure to dehydroPAs is involved deserves further investigation, especially if other features of dehydroPA poisoning are also present such as hypertrophic (megalocytic) cells, antimitotic effects and evidence of immunotoxicity.

6. CONCLUSIONS AND FUTURE DIRECTIONS Recent recognition that some individuals and communities may be intermittently exposed to hazardous levels of mutagenic dehydroPAs, naturally present or present as contaminants in a number of common foods, raises the possibility that these natural toxins may be important contributors to the development of certain chronic diseases in those individuals and communities. However epidemiological support is required and considerable research is needed to establish the veracity and extent of this possibility. As well as increased regional analyses of food to determine current levels of dietary exposure to dehydroPAs, awareness amongst clinicians of this possibility should lead to questioning of patients about their dietary habits and the sources of the foods they consume. The chronic diseases that could sometimes be caused, initiated or precipitated by the current intermittent, low levels of dietary exposure to dehydroPAs include: a) a wide range of cancers; b) PAH; c) progressive liver disease leading to cirrhosis; and, d) congenital anomalies, or a combination of these. PAH and HSOS in particular, are highly characteristic 23



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and indicative responses to dehydroPA exposure. All cases of chronic illness involving or accompanied by evidence of either or both of these relatively rarely co-occurring conditions could indicate a possible dietary dehydroPA etiology.160,207 The availability of methods for detecting and quantifying DHP-DNA71,261, and DHPprotein adducts262 provides means of measuring levels of dietary exposure to dehydroPAs in different populations and allow the level of DHP adducts detected to be correlated with the incidence of certain chronic diseases. Such analysis is, for example, particularly called for in regions where honeys from dehydroPA-producing plants are harvested and sold locally or when flour from locally harvested grain could sometimes be contaminated. These data can then be correlated with the relative incidence of associated chronic diseases in these particular regions. The current animal models of PAH produced by exposure to dehydroPAs are aimed at generating high numbers of affected animals relatively quickly for experimental purposes and some acute effects of dehydroPAs are therefore also produced. While these animal models do indeed reflect all of the important aspects of the pathophysiology of PAH, they do not mimic likely low level and intermittent dietary exposure to these natural toxins. There is a need to explore these animal models using levels of dehydroPAs equivalent to those expected from current levels and patterns of food contamination in at-risk communities. Congenital anomalies in fetuses and neonates, caused by dehydroPA at levels equivalent to those currently occurring in the food supply, also need to be explored in animal models as does the apparent role of defective BMP signaling as a common mechanism in, and cause of, several possible dehydroPA-associated diseases.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

Funding Sources

None

Conflict of interest

None

ACKNOWLEDGEMENTS

None

ABBREVIATIONS

HSOS, hepatic sinusoidal obstruction syndrome; dehydroPAs, 1,2-dehydropyrrolizidine ester alkaloids; PAH, pulmonary arterial hypertension; bw, body weight; CYP450, cytochrome P450 monoamine oxidases; DHP, 1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine; GSH, glutathione; MMPs, metalloproteinases; POPH, portopulmonary hypertension; BMPR2, bone morphogenic protein receptor II; VODI, hepatic veno-occlusive disease with immunodeficiency; RMS, rhabdomyosarcoma; SCT, allogeneic stem cell transplantation; MIOP, malignant infantile osteopetrosis; PID, primary immunodeficiency; SIDs, severe immune dysregulatory disorders; FOP, fibrodysplasia ossification progressive; BWS, Beckwith-Wiedemann Syndrome.

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Figure Captions 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. Figure 2. The three most common necine bases defining dehydroPAs, viz. retronecine, heliotridine and otonecine; a general structure representing dehydroPA N-oxides; and structures representing the four main categories of dehydroPA pro-toxins. Figure 3. Liver metabolism of dehydroPAs, using the macrocyclic diester riddelliine as an example. Figure 4. A typical mixture of DHP adducts produced by reaction of galectin-1 with dehydromonocrotaline.112

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A B

C

D

Figure 1

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Figure 2 58


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Figure 3

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Figure 4

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