Perspective

pubs.acs.org/crt

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
1. Introduction 4
Notes
Abbreviations
13
13
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
fragments are removed. However, if dehydroPA-containing
plants were present in the crop, then dust containing
dehydroPAs is not removed by screening and, consequently,
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
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
production, 41 and the honey produced is significantly
5 DOI: 10.1021/tx500403t
contaminated by dehydroPAs, e.g., up to 2000−4000 μg 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.18,31,32,42−44 However, based on a consid-
eration of their well-established genotoxicity8,45,46 and on
recent risk assessments suggesting that to make cancer unlikely
a maximum tolerable daily intake of 0.007 μg 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 failure,8,20,47 and may 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.
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
Many cases of cancer, PAH, congenital anomalies, and liver
disease are of unknown etiology, so establishing that 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.
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 Approx-
imately 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)
Figure 2. Three most common necine bases defining dehydroPAs, viz.,
retronecine, heliotridine, and otonecine; a general structure represent-
ing dehydroPA N-oxides; and structures representing the four main
categories of dehydroPA pro-toxins.
6 DOI: 10.1021/tx500403t
Perspective
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 pro-toxins. 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 bifunc-
tional biological alkylating agents capable of causing tissue
damage and inducing genetic mutations.52 In vivo N-oxidation
of dehydroPAs by CYP450 enzymes and flavin-containing
mono-oxygenases68 is a detoxifying mechanism that leads 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
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
dehydroPAs, is carcinogenic104−106 and immunosuppressive107
and mimics the antimitotic effects of radiation in tissues
displaying high rates of mitosis.108−111 Circulating DHP is
considered to be responsible for the effects of dehydroPAs
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
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
DHP that remains unadducted 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
monoadducted, 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.52,95−97,109−111,113 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.
7 DOI: 10.1021/tx500403t
Perspective
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 per kilogram of bodyweight,
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
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 dis-
eases.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
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
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
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
per kilogram of bodyweight per 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.
3.2. Pulmonary Arterial Hypertension (PAH). Recog-
nition 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
animals,56,126−133 and, since then, dehydroPAs such as
monocrotaline and fulvine have been deliberately administered
by medical researchers to various animal species to generate
8 DOI: 10.1021/tx500403t
Perspective
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 nonhuman 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 disease,131,132,136 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
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
expected to slowly produce PAH more typical of the disease
normally seen in humans and in the nonhuman primate model
developed by Chesney and Allen.130−132
Several experiments with nonhuman 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, whereas 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 (dehydro-
monocrotaline) 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 microgram per kilogram of bodyweight 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 short-lived 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 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, suggest 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 exper-
imental 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 ani-
mals.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 Li-Fraumeni syndrome caused by
germline mutations of TP53,147 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
9 DOI: 10.1021/tx500403t
Perspective
dehydroPA N-oxide, indicine-N-oxide, was at one time clinically
investigated for use as a cancer chemotherapeutic agent.148,149
The antimitotic 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 develop-
ment 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, as this type of exposure
allows intervening periods 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 dehy-
droPAs 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 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 Treat-
ment 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 HSOS, 178−180 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
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
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, then 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 ability of some dehydroPA-affected livers to biosynthesize
GSH and thus dehydroPA-exposed 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
intraperitoneal 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 heteroge-
neous 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 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
10
Perspective
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 PAH,185−187 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, hyper-
trophic (megalocytic), apoptosis-resistant cells, in particular
smooth muscle cells, that contribute to the characteristic
plexiform (onionskin-like) 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 quasi-malignant 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 kidney,4,152 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 to be incapable of division.196,197 This
characteristic is, however, believed to be due to the persistent
antimitotic effects of dehydroPAs, and, given 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 environ-
mental factors and further somatic mutations may be needed
before PAH develops in people carrying BMPR2 mutations. It
has therefore been suggested that a so-called 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 but also could 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 hyper-
tension.202−204 The exact pathogenesis and etiology of POPH
are poorly understood.205 One clinical study of 2459 patients
with cirrhosis showed that 15 (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
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
condition sometimes caused by dehydroPAs in animals, so
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 suggest 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 it is not surprising that ineffective bone formation is
among 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, then 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 years) 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
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 as well
as the susceptibility to developing certain cancers, including
RMS, and concurrent susceptibility to HSOS during chemo-
therapy 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, especially those showing evidence of HSOS. While
clinicians dealing with cases of HSOS, especially in neonates
and young children, often question 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.
11
Perspective
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 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 dehydroPAs240,241 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 a nonfunctional 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 commod-
ities such as grain, milk, and honey for wide national and
international distribution leads to dilution of dehydroPA
contaminants and ensures that dietary exposure to acutely
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
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., poly-
chlorinated biphenyls, induce cytochrome CYP3A enzymes, so
concurrent consumption of or exposure to these could increase
an individual’s susceptibility to dehydroPA toxicity via
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
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 dehydroPA,52,130 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 herb-
al 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 1 μg,
reduced to 0.1 μg if the medication is taken daily for more
than 6 weeks per year.257 These regulations are not currently
applied to food, although a significant number of retail food
items have been shown to exceed the maximum levels of
dehydroPAs allowed in phytopharmaceuticals in Ger-
many.21,27−29,33−40
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 that may be
associated with dietary exposure to dehydroPAs. Until this is
achieved, the following indicators, rationally based on
associative considerations described above, are recommended
as possible indications of a dietary dehydroPA etiology and
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 the six indicators comprising the
above list, several cases of liver disease and cancers with a
p o s s i b l e d e h ydr o P A e t i o l o g y ha v e b e e n iden t i -
fied.158−160,163,168,176,222−226,231−234,236−238,245,247−249,259,260 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, swel-
ling),222,232,233,237,238 inconsistent veno-occlusive disease
12
Perspective
(HSOS),238 and rare mitotic figures223 that are indicative of a
dehydroPA etiology.
If several of the indicators suggested above are present in an
individual case, then 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 develop-
ment 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 among 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
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 DHP−protein adducts262 provides
means of measuring levels of dietary exposure to dehydroPAs
in different populations and allows 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.
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +61 2 9490 5000; Fax: +61 2 9490 8499; E-mail: john.
edgar@csiro.au.
Notes
The authors declare no competing financial interest.
■ 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, metallopro-
teinases; 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
■
REFERENCES
(1) Willmot, F. C., and Robertson, G. W. (1920) Senecio disease, or
cirrhosis of the liver due to Senecio poisoning. Lancet, 848−849.
(2) Steyn, D. G. (1933) Poisoning of human beings by weeds
contained in cereals (Bread Poisoning). Onderstepoort J. Vet. Sci. Anim.
Ind. 1, 219−266.
(3) Selzer, G., and Parker, R. G. F. (1951) Senecio poisoning as
Chiari’s syndrome. A report on twelve cases. Am. J. Pathol. 27, 885−
907.
(4) Bull, L. B., Culvenor, C. C. J., and Dick, A. J. (1968) The
Pyrrolizidine Alkaloids. Their Chemistry, Pathogenicity and Other
Biological Properties, North Holland Publishing Co., Amsterdam, The
Netherlands.
(5) Tandon, B. N., Tandon, R. K., Tandon, H. D., Narndranathan,
M., and Joshi, Y. K. (1976) An epidemic of veno-occlusive disease of
liver in central India. Lancet 2, 271−272.
(6) Tandon, R. K., Tandon, B. N., Tandon, H. D., Bhatia, M. L.,
Bhargava, S., Lal, P., and Arora, R. R. (1976) Study of an epidemic of
venoocclusive disease in India. Gut 17, 849−855.
(7) Mohabbat, O., Younos, M. S., Merzad, A. A., Srivastava, R. N.,
Sediq, G. G., and Aram, G. N. (1976) An outbreak of hepatic veno-
occlusive disease in North-Western Afghanistan. Lancet 308, 269−271.
(8) International Programme on Chemical Safety (IPCS) (1988)
Pyrrolizidine alkaloids: Environmental Health Criteria 80, WHO,
Geneva, Switzerland, www.inchem.org/documents/ehc/ehc/ehc080.
htm.
(9) International Programme on Chemical Safety (IPCS) (1989)
Pyrrolizidine alkaloids: health and safety guide no. 26; WHO, Geneva,
Switzerland, http://www.inchem.org/documents/hsg/hsg/hsg026.
htm.
(10) Chauvin, P., Dillon, J., Moren, A., Talbak, S., and Barakaev, S.
(1993) Heliotrope poisoning in Tadjikistan. Lancet 341, 1663.
(11) Chauvin, P., Dillon, J. C., and Moren, A. (1994) An outbreak of
heliotrope food poisoning, Tadjikistan, November 1992−March 1993.
Sante 4, 263−268.
(12) Mayer, F., and Lüthy, J. (1993) Heliotrope poisoning in
Tadjikistan. Lancet 342, 246−247.
(13) Altaee, M. Y., and Mahmood, M. H. (1998) An outbreak of
veno-occlusive disease of the liver in northern Iraq. East. Mediterr.
Health J. 4, 142−148.
(14) Integrated Regional Information Networks (IRIN) (2008)
Afghanistan: “Charmak” disease still killing people, livestock in west, UN
Office for the Coordination of Humanitarian Affairs, http://www.
irinnews.org/Report.aspx?ReportId=81971.
13
Perspective
(15) Kleiman, R., Rentz, E. D., Teshale, E., Thompson, N., and
Schurz-Rogers, H. (2008) Update on research and activities at the
Centers for Disease Control and Prevention, and the Agency for Toxic
Substances and Disease Registry. J. Med. Toxicol. 4, 197−200.
(16) Kakar, F., Akbarian, Z., Leslie, T., Mustafa, M. L., Watson, J., van
Egmond, H. P., Omar, M. F., and Mofleh, J. (2010) An outbreak of
hepatic veno-occlusive disease in western Afghanistan associated with
exposure to wheat flour contaminated with pyrrolizidine alkaloids. J.
Toxicol. 2010, 313280.
(17) Edgar, J. A. (2003) Pyrrolizidine alkaloids and food safety.
Chem. Aust. 70, 4−7.
(18) Food Standards Australia New Zealand (FSANZ) (2001)
Pyrrolizidine alkaloids in food. A toxicological review and risk assessment,
Tech. Report series No. 2, FSANZ, Canberra, Australia, http://www.
foodstandards.gov.au/publications/documents/TR2.pdf.
(19) Azadbakht, M., and Talavaki, M. (2003) Qualitative and
quantitative determination of pyrrolizidine alkaloids of wheat and flour
contaminated with Senecio in Mazandaran Province farms. Iran. J.
Pharm. Res. 2, 179−183.
(20) Edgar, J. A., Colegate, S. M., Boppré, M., and Molyneux, R. J.
(2011) Pyrrolizidine alkaloids in food: a spectrum of potential health
consequences. Food Addit. Contam. 28, 308−324.
(21) Bodi, D., Ronczka, S., Gottschalk, C., Behr, N., Skibba, A.,
Wagner, M., Lahrssen-Wiederholt, M., Preiss-Weigert, A., and These,
A. (2014) Determination of pyrrolizidine alkaloids in tea, herbal drugs
and honey. Food Addit. Contam. 31, 1886−1895.
(22) Dickinson, J. O., and King, R. R. (1978) The transfer of
pyrrolizidine alkaloids from Senecio jacobaea into milk of lactating cows
and goats, in Effects of Poisonous Plants on Livestock (Keeler, I.,
VanKampen, K. R., and James, L. F., Eds.) pp 201−206, Academic
Press, New York.
(23) Deinzer, M. L., Thomson, P. A., Burgett, D. M., and Isaacson, D.
L. (1977) Pyrrolizidine alkaloids: their occurrence in honey from tansy
ragwort (Senecio jacobaea L.). Science 195, 497−499.
(24) Culvenor, C. C. J., Edgar, J. A., and Smith, L. W. (1981)
Pyrrolizidine alkaloids in honey from Echium plantagineum L. J. Agric.
Food Chem. 29, 958−960.
(25) Lüthy, J., Heim, T., and Schlatter, C. (1983) Transfer of
[3H]pyrrolizidine alkaloids from Senecio vulgaris L. and metabolites
into rat milk and tissues. Toxicol. Lett. 17, 283−288.
(26) Molyneux, R. J., and James, L. F. (1990) Pyrrolizidine alkaloids
in milk: thresholds of in toxication. Vet. Hum. Toxicol. 32 (Suppl.),
94S−103S.
(27) Edgar, J. A., and Smith, L. W. (2000) Transfer of pyrrolizidine
alkaloids into eggs: food safety implications, in Natural and Selected
Synthetic Toxins, Biological Implications. (Tu, A. T., and Gaffield, W.,
Eds.) ACS Symposium Series 745, Chapter 8, pp 118−128, American
Chemical Society, Washington, DC.
(28) Beales, K. A., Betteridge, K., Colegate, S. M., and Edgar, J. A.
(2004) Solid phase extraction and LCMS analysis of pyrrolizidine
alkaloids in honeys. J. Agric. Food Chem. 52, 6664−6672.
(29) Beales, K, Betteridge, K, Boppré, M, Cao, Y, Colegate, S. M.,
Edgar, J. A. (2007) Hepatotoxic pyrrolizidine alkaloids and their N-
oxides in honey and pollen, in Poisonous Plants: Global Research and
Solutions (Panter, K., Wierenga, T., and Pfister, J., Eds.) Chapter 16, pp
94−100, CABI, Wallingford.
(30) Bundesamt für Risiokobewertung (BfR) (2007) Salatmischung
mit pyrrolizidinalkaloid-haltigem Greiskraut verunreinigt, Stellungnahme
Nr.028/2007 des BfR vom 10, http://www.bfr.bund.de/cm/208/
salatmischung_mit_pyrrolizidinalkaloid_haltigem_geiskraut_
verunreinigt.pdf.
(31) Bundesamt für Risiokobewertung (BfR) (2011) Chemical
analysis and toxicity of pyrrolizidine alkaloids and assessment of the
health risk posed by their occurrence in honey, BfR opinion no. 038/2011
11, http://www.bfr.bund.de/cm/349/chemical-analysis-and-toxicity-
of-pyrrolizidine-alkaloids-and-assessment-of-the-health-risks-posed-by-
their-occurence-in-honey.pdf.
(32) Bundesamt für Risiokobewertung (BfR) (2013) Pyrrolizidine
alkaloids in herbal teas and teas, BfR opinion no. 018/2013, http://
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
www.bfr.bund.de/cm/349/pyrrolizidine-alkaloids-in-herbal-teas-and-
teas.pdf.
(33) Kempf, M., Heil, S., Hasslauer, I., Schmidt, L., von der Ohe, K.,
Theuring, C., Reinhard, A., Scheier, P., and Beuerle, T. (2010)
Pyrrolizidine alkaloids in pollen and pollen products. Mol. Nutr. Food
Res. 54, 1−9.
(34) Hoogenboom, L. A. P., Mulder, P. P. J., Zeilmaker, M. J., van
den Top, H. J., Remmelink, G. J., Brandon, E. F. A., Klijnstra, M.,
Meijer, G. A. L., Schothorst, R., and van Egmond, H. P. (2011) Carry-
over of pyrrolizidine alkaloids from feed in dairy cows. Food Addit.
Contam. 28, 359−372.
(35) Dübecke, A., Beckh, G., and Lüllmann, C. (2011) Pyrrolizidine
alkaloids in honey and bee pollen. Food Addit. Contam. 28, 348−358.
(36) Fletcher, M. T., McKenzie, R. A., Reichmann, K. G., and Blaney,
B. J. (2011) Risks from plants containing pyrrolizidine alkaloids for
livestock and meat quality in northern Australia, in Poisonings by Plants,
Mycotoxins and Related Toxins (Riet-Correa, F., Pfister, J., Schild, A. L.,
and Wierenga, T., Eds.) pp 208−218, CAB International, Wallingford,
UK.
(37) Kempf, M., Wittig, M., Schönfeld, K., Cramer, L., Schreier, P.,
and Beuerle, T. (2011) Pyrrolizidine alkaloids in food: downstream
contamination in the food chain caused by honey and pollen. Food
Addit. Contam. 28, 325−331.
(38) Kempf, M., Wittig, M., Reinhard, A., von der Ohe, K.,
Blacquiére, T., Raezke, K.-P., Michel, R., Scheier, P., and Beuerle, T.
(2011) Pyrrolizidine alkaloids in honey: comparison of analytical
methods. Food Addit. Contam. 28, 332−347.
(39) Cao, Y., Colegate, S. M., and Edgar, J. A. (2013) Persistence of
echimidine, a hepatotoxic pyrrolizidine alkaloid, from honey into
mead. J. Food Compos. Anal. 29, 106−109.
(40) Griffin, C. T., Danaher, M., Elliott, C. T., Kennedy, D. G., and
Furey, A. (2013) Detection of pyrrolizidine alkaloids in commercial
honey using liquid chromatography-ion trap mass spectrometry. Food
Chem. 136, 1577−1583.
(41) Edgar, J. A., Roeder, E., and Molyneux, R. J. (2002) Honey from
plants containing pyrrolizidine alkaloids: a potential threat to health. J.
Agric. Food Chem. 50, 2719−2730.
(42) Committee on Toxicity of Chemicals in Food, Consumer
Products and the Environment (COT) (2008) COT statement on
pyrrolizidine alkaloids in food, http://cot.food.gov.uk/pdfs/
cotstatementpa200806.pdf.
(43) WHO (2011) Discussion paper on pyrrolizidine alkaloids, Joint
FAO/WHO food standards programme, Codex committee on
contaminants in foods, 5th session, The Hague, The Netherlands,
21−25 March, ftp://ftp.fao.org/codex/meetings/cccf/cccf5/cf05_14e.
pdf.
(44) European Food Safety Authority (EFSA) Panel on
Contaminants in the Food Chain (CONTAM) (2011) Scientific
opinion on pyrrolizidine alkaloids in food and feed. EFSA J. 9, 2406−
2540.
(45) The U.S. National Toxicology Program (2011) 12th Report on
Carcinogens (RoC), pp iii−499, U.S. Department of Health and Human
Services, Washington, DC.
(46) Chen, T., Mei, N., and Fu, P. P. (2010) Genotoxicity of
pyrrolizidine alkaloids. J. Appl. Toxicol. 30, 183−196.
(47) Edgar, J. A. (2014) Food contaminants capable of causing
cancer, pulmonary hypertension and cirrhosis. Med. J. Aust. 200, 73−
74.
(48) Green, C. R., and Christie, G. S. (1961) Malformation in foetal
rats induced by the pyrrolizidine alkaloid, heliotrine. Br. J. Exp. Pathol.
42, 369−378.
(49) Peterson, J. E., and Jago, M. V. (1980) Comparison of the toxic
effects of dehydroheliotridine and heliotrine in pregnant rats and their
embryos. J. Pathol. 131, 339−355.
(50) Smith, L. W., and Culvenor, C. C. J. (1981) Plant sources of
hepatotoxic pyrrolizidine alkaloids. J. Nat. Prod. 44, 129−152.
(51) Reimann, A., Nurhayati, N., Backenköhler, A., and Ober, D.
(2004) Repeated evolution of the pyrrolizidine alkaloid-mediated
14
Perspective
defense system in separate angiosperm lineages. Plant Cell 16, 2772−
2784.
(52) Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine
Alkaloids, Academic Press, London, UK.
(53) Mattocks, A. R. (1968) Toxicity of pyrrolizidine alkaloids.
Nature 217, 723−728.
(54) Culvenor, C. C. J., Downing, D. T., Edgar, J. A., and Jago, M. V.
(1969) Pyrrolizidine alkaloids as alkylating and antimitotic agents.
Ann. N.Y. Acad. Sci. 163, 837−847.
(55) Jago, M. V., Edgar, J. A., Smith, L. W., and Culvenor, C. C. J.
(1970) Metabolic conversion of heliotridine-based pyrrolizidine
alkaloids to dehydroheliotridine. Mol. Pharmacol. 6, 402−406.
(56) Butler, W. H., Mattocks, A. R., and Barnes, J. M. (1970) Lesions
in the liver and lungs of rats given pyrrole derivatives of pyrrolizidine
alkaloids. J. Pathol. 100, 169−175.
(57) Williams, D. E., Reed, R. L., Kedzierski, B., Dannan, G. A.,
Guengerich, F. P., and Buhler, D. R. (1989) Bioactivation and
detoxication of the pyrrolizidine alkaloid senecionine by cytochrome
P-450 enzymes in rat liver. Drug Metab. Dispos. 17, 387−392.
(58) Miranda, C. L., Reed, R. L., Guengerich, F. P., and Buhler, D. R.
(1991) Role of cytochrome P450IIIA4 in the metabolism of the
pyrrolizidine alkaloid senecionine in human liver. Carcinogenesis 12,
515−519.
(59) Hincks, J. R., Kim, H.-Y., Segall, H. J., Molyneux, R. J., Stermitz,
F. R., and Coulombe, R. A. (1991) DNA cross-linking in mammalian
cells by pyrrolizidine alkaloids: structure−activity relationships.
Toxicol. Appl. Pharmacol. 111, 90−98.
(60) Chung, W. G., and Buhler, D. R. (1994) The effect of
spironolactone treatment on the cytochrom P450-mediated metabo-
lism of the pyrrolizidine alkaloid senecionine by hepatic microsomes
from rats and guinea pigs. Toxicol. Appl. Pharmacol. 127, 314−319.
(61) Chung, W. G., Miranda, C. L., and Buhler, D. R. (1995) A
cytochrome P450B form is the major bioactivation enzyme for the
pyrrolizidine alkaloid senecionine in guinea pig. Xenobiotica 25, 929−
939.
(62) Kim, H.-Y., Stermitz, F. R., and Coulombe, R. A. (1995)
Pyrrolizidine alkaloid-induced DNA-protein cross-links. Carcinogenesis
16, 2691−2697.
(63) Kasahara, Y., Kiyatake, K., Tatsumi, K., Sugito, K., Kakusaka, I.,
Yamagata, S., Ohmori, S., Kitada, M., and Kuriyama, T. (1997)
Bioactivation of monocrotaline by P450 3A in rat liver. J. Cardiovasc.
Pharmacol. 30, 124−129.
(64) Reid, M. J., Lamé, M. W., Morin, D., Wilson, D. W., and Segall,
H. J. (1998) Involvement of cytochrome P450 3A in the metabolism
and covalent binding of 14C-monocrotaline in rat liver microsomes. J.
Biochem. Mol. Toxicol. 12, 157−166.
(65) Pereira, T. N., Webb, R. I., Reilly, P. E. B., Seawright, A. A., and
Prakash, A. S. (1998) Dehydromonocrotaline generates sequence-
selective N-7 guanine alkylation and heat and alkali stable multiple
fragment DNA crosslinks. Nucl. Acid Res. 26, 5441−5447.
(66) Tepe, J. J., and Williams, R. M. (1999) DNA cross-linking by a
phototriggered dehydromonocrotaline progenitor. J. Am. Chem. Soc.
121, 2951−2955.
(67) Lin, G., Cui, Y. Y., and Hawes, E. M. (2000) Characterization of
rat liver microsomal metabolites of clivorine, an hepatotoxic
otonecine-type pyrrolizidine alkaloid. Drug Metab. Dispos. 28, 1475−
1483.
(68) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2004) Pyrrolizidine
alkaloidsgenotoxicity, metabolism enzymes, metabolic activation,
and mechanisms. Drug Metab. Rev. 36, 1−55.
(69) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2002) Genotoxic
pyrrolizidine alkaloidsmechanism leading to DNA adduct formation
and tumorigenicity. Int. J. Mol. Sci. 3, 948−964.
(70) Yang, Y.-C., Yan, J., Doerge, D. R., Chan, P.-C., Fu, P. P., and
Chou, M. W. (2001) Metabolic activation of the tumorigenic
pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation
in vivo. Chem. Res. Toxicol. 14, 101−109.
(71) Yang, Y.-C., Yan, J., Churchwell, M., Beger, R., Chan, P.-C.,
Doerge, D. R., Fu, P. P., and Chou, M. W. (2001) Development of a
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
32
P-postlabeling/HPLC method for detection of dehydroretronecine-
derived DNA adducts in vivo and in vitro. Chem. Res. Toxicol. 14, 91−
100.
(72) Xia, Q., Chou, M. W., Kadlubar, F. F., Chan, P., and Fu, P. P.
(2003) Human liver microsomal metabolism and DNA adduct
formation of the tumorigenic pyrrolizidine alkaloid, riddelliine.
Chem. Res. Toxicol. 16, 66−73.
(73) Chou, M. W., Yan, J., Nichols, J., Xia, Q., Beland, F. A., Chan, P.
C., and Fu, P. P. (2003) Correlation of DNA adduct formation and
riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice.
Cancer Lett. 193, 119−125.
(74) Xia, Q., Chou, M. W., Lin, G., and Fu, P. P. (2004) Metabolic
formation of DHP-derived DNA adducts from a representative
otonecine type pyrrolizidine alkaloid clivorine and the extract of
Ligularia hodgsonnii Hook. Chem. Res. Toxicol. 17, 702−708.
(75) Xia, Q., Chou, M. W., Edgar, J. A., Doerge, D. R., and Fu, P. P.
(2006) Formation of DHP-derived DNA adducts from metabolic
activation of the prototype heliotridine-type pyrrolizidine alkaloid,
lasiocarpine. Cancer Lett. 231, 138−145.
(76) Mattocks, A. R., and Bird, I. (1983) Pyrrolic and N-oxide
metabolites formed from pyrrolizidine alkaloids by hepatic micro-
somes in vitrorelevance to in vivo hepatotoxicity. Chem.−Biol.
Interact. 43, 209−222.
(77) Bull, L. B., and Dick, A. T. (1959) The chronic pathological
effects on the liver of the rat of the pyrrolizidine alkaloids heliotrine,
lasiocarpine and their N-oxides. J. Pathol. Bacteriol. 78, 483−502.
(78) Mattocks, A. R. (1971) Hepatotoxic effects due to pyrrolizidine
alkaloid N-oxides. Xenobiotica 1, 563−565.
(79) Molyneux, R. J., Johnson, A. E., Olson, J. D., and Baker, D. C.
(1991) Toxicity of pyrrolizidine alkaloids from Riddell groundsel
(Senecio riddellii) to cattle. Am. J. Vet. Res. 52, 146−151.
(80) Chou, M. W., Wang, J. Y., Yang, Y.-C., Beger, R. D., Williams, L.
D., Doerge, D. R., and Fu, P. P. (2003) Riddelliine N-oxide is a
phytochemical and mammalian metabolite with genotoxic activity that
is comparable to the parent pyrrolizidine alkaloid riddelliine. Toxicol.
Lett. 145, 239−247.
(81) Wang, Y.-P., Yan, J., Fu, P. P., and Chou, M. W. (2005) Human
liver microsomal reduction of pyrrolizidine alkaloid N-oxides to form
the corresponding carcinogenic parent alkaloid. Toxicol. Lett. 155,
411−420.
(82) Yan, J., Xia, Q., Chou, M. W., and Fu, P. P. (2008) Metabolic
activation of retronecine and retronecine-N-oxideformation of
DHP-derived DNA adducts. Toxicol. Ind. Health 24, 181−188.
(83) Cao, Y., Colegate, S. M., and Edgar, J. A. (2008) Safety
assessment of food and herbal products containing hepatotoxic
pyrrolizidine alkaloids: inter-laboratory consistency and the impor-
tance of N-oxide determination. Phytochem. Anal. 19, 526−533.
(84) Mattocks, A. R. (1968) Iron (II)-catalysed conversion of
unsaturated pyrrolizidine alkaloid N-oxides to pyrrole derivatives.
Nature 219, 480.
(85) Culvenor, C. C. J., Edgar, J. A., Smith, L. W., and Tweedale, H. J.
(1969) Dihydropyrrolizine analogues of pyrrolizidine alkaloids. Aust. J.
Chem. 41, 3599−3602.
(86) Mattocks, A. R. (1969) Dihydropyrrolizine derivatives from
unsaturated pyrrolizidine alkaloids. J. Chem. Soc. C, 1155−1162.
(87) Culvenor, C. C. J., Edgar, J. A., Smith, L. W., and Tweedale, H. J.
(1970) Dihydropyrrolizines III. Preparation and reactions of
derivatives related to pyrrolizidine alkaloids. Aust. J. Chem. 23,
1853−1867.
(88) Mattocks, A. R., and Jukes, R. (1990) Trapping and
measurement of short-lived alkylating agents in a recirculating flow
system. Chem.−Biol. Interact. 76, 19−30.
(89) Mattocks, A. R., Driver, H. E., Barbour, R. H., and Robins, D. J.
(1986) Metabolism and toxicity of synthetic analogues of macrocyclic
diester pyrrolizidine alkaloids. Chem.−Biol. Interact. 58, 95−108.
(90) Eastman, D. F., Dimenna, G. P., and Segall, H. J. (1982)
Covalent binding of two pyrrolizidine alkaloids, senecionine and
seneciphylline, to hepatic macromolecules and their distribution,
15
Perspective
excretion, and transfer into milk of lactating mice. Drug Metab. Dispos.
10, 236−240.
(91) Chou, M. W., and Fu, P. P. (2006) Formation of DHP-derived
DNA adducts in vivo from dietary supplements and Chinese herbal
plant extracts containing carcinogenic pyrrolizidine alkaloids. Toxicol.
Ind. Health 22, 321−327.
(92) Hsu, I. C., Allen, J. R., and Chesney, C. F. (1973) Identification
and toxicological effects of dehydroretronecinemetabolite of
monocrotaline. Proc. Soc. Exp. Biol. Med. 144, 834−838.
(93) Hsu, I. C., Robertson, K. A., Shumaker, R. C., and Allen, J. R.
(1975) Binding of tritiated dehydroretronecine to macromolecules.
Res. Commun. Chem. Pathol. Pharmacol. 11, 99−106.
(94) Curtain, C. C., and Edgar, J. A. (1976) The binding of
dehydroheliotridine to DNA and the effect of it and other compounds
on repair synthesis in main and satellite band DNA. Chem.−Biol.
Interact. 13, 243−256.
(95) Sun, P. S., Hsia, M-T. S., Chu, F. S., and Allen, J. R. (1977)
Inhibition of yeast alcohol dehydrogenase by dehydroretronecine.
Food Cosmet. Toxicol. 15, 419−422.
(96) Seawright, A. A. (1992) Potential toxicity problems with herbal
medicines and foodnew observations with pyrrolizidine alkaloids.
Proc. Nutr. Soc. Aust. 17, 20−25.
(97) Seawright, A. A. (1994) Toxic plant residues in meat, in Plant-
Associated Toxins; Agricultural, Phytochemical and Ecological Aspects
(Colegate, S. M., and Dorling, P. R., Eds.) Chapter 15, pp 77−84, CAB
International, Wallingford UK.
(98) Hsu, I. C., Schumaker, R. C., and Allen, J. R. (1974) Tissue
distribution of tritium-labeled dehydroretronecine. Chem.−Biol.
Interact. 8, 163−170.
(99) Hsu, I. C., Robertson, K. A., and Allen, J. R. (1976) Tissue
distribution, binding properties and lesions produced by dehydrore-
tronecine in the non-human primate. Chem.−Biol. Interact. 12, 19−28.
(100) Shumaker, R. C., Hsu, I. C., and Allen, J. R. (1976)
Localisation and tissue effects of tritiated dehydroretronecine in young
rats. J. Pathol. 119, 21−28.
(101) Mattocks, A. R., and White, I. N. H. (1976) The distribution of
[3H]synthanecine a bis-N-ethylcarbamate and its metabolites in the
rat. Chem.−Biol. Interact. 15, 173−184.
(102) Candrian, U., Lüthy, J., and Schlatter, C. (1985) In vivo
covalent binding of retronecine-labeled [3H] seneciphylline and [3H]
senecionine to DNA of rat liver, lung and kidney. Chem.−Biol. Interact.
54, 57−69.
(103) Mattocks, A. R., and Jukes, R. (1990) Recovery of the pyrrolic
nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates
in tissues and body fluids. Chem.−Biol. Interact. 75, 225−239.
(104) Allen, J. R., Hsu, I.-C., and Carstens, L. A. (1975)
Dehydroretronecine induced rhabdomyosarcomas in rats. Cancer Res.
35, 997−1002.
(105) Shumaker, R. C., Robertson, K. A., Hsu, I. C., and Allen, J. R.
(1976) Neoplastic transformation in tissues of rats exposed to
monocrotaline or dehydroretronecine. J. Natl. Cancer Inst. 56, 787−
789.
(106) Peterson, J. E., Jago, M. V., Reddy, J. K., and Jarrett, R. G.
(1983) Neoplasia and chronic disease associated with the prolonged
administration of dehydroheliotridine to rats. J. Natl. Cancer Inst. 70,
381−386.
(107) Percy, J., and Pierce, A. E. (1971) Immunosuppressive activity
of the pyrrolizidine alkaloid metabolite dehydroheliotridine. Immunol-
ogy 21, 273−280.
(108) Peterson, J. E., Samuel, A., and Jago, M. V. (1972) Pathological
effects of dehydroheliotridine, a metabolite of heliotridine-based
pyrrolizidine alkaloids in the young rat. J. Pathol. 107, 175−189.
(109) Mattocks, A. R., and Bird, I. (1983) Alkylation by
dehydroretronecine, a cytotoxic metabolite of some pyrrolizidine
alkaloids: an in vivo test. Toxicol. Lett. 16, 1−8.
(110) Prakash, A. S., Pereira, T. N., Reilly, P. E. B., and Seawright, A.
A. (1999) Pyrrolizidine alkaloids in human diet. Mutat. Res. 443, 53−
67.
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
(111) Hooper, P. T. (1975) Experimental acute gastrointestinal
disease caused by the pyrrolizidine alkaloid, lasiocarpine. J. Comp.
Pathol. 85, 341−349.
(112) Wong, L. S.N., Lamé, M. W., Jones, A. D., and Wilson, D. W.
(2010) Differential cellular responses to protein adducts of
naphthoquinone and monocrotaline pyrrole. Chem. Res. Toxicol. 23,
1504−1513.
(113) Molyneux, R. J., Johnson, A. E., and Stuart, L. D. (1988)
Delayed manifestation of Senecio-induced pyrrolizidine alkaloidosis in
cattle: case reports. Vet. Hum. Toxicol. 30, 201−205.
(114) DeLeve, L. D., Wang, X., Kuhlenkamp, J. F., and Kaplowitz, N.
(1996) Toxicity of azathioprine and monocrotaline in murine
sinusoidal endothelial cells and hepatocytes: the role of glutathione
and relevance to hepatic venooclusive disease. Hepatology 23, 589−
599.
(115) DeLeve, L. D., McCuskey, R. S., Wang, X., Hu, L., McCuskey,
M. K., Epstein, R. B., and Kanel, G. C. (1999) Characterization of a
reproducible rat model of hepatic veno-occlusive disease. Hepatology
29, 1779−1791.
(116) DeLeve, L. D., Shulman, H. M., and McDonald, G. B. (2002)
Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome
(veno-occlusive disease). Semin. Liver Dis. 22, 27−41.
(117) DeLeve, L. D., Ito, Y., Bethea, N. W., McCuskey, M. K., Wang,
X., and McCuskey, R. S. (2003) Embolization by sinusoidal lining cells
obstructs the microcirculation in rat sinusoidal obstruction syndrome.
Am. J. Physiol.: Gastrointest. Liver Physiol. 284, G1045−G1052.
(118) DeLeve, L. D., Valla, D.-C., and Garcia-Tsao, G. (2009)
Vascular disorders of the liver. Hepatology 49, 1729−1764.
(119) Huxtable, R. J. (1989) Human health implications of
pyrrolizidine alkaloids and herbs containing them, in Toxicants of
Plant Origin (Cheeke, P. R., Ed). Vol. 1, Chapter 3, pp 42−86, CRC
Press, Inc., Boca Raton, FL.
(120) Nolan, J. P., Scheig, R. L., and Klatskin, G. (1966) Delayed
hepatitis and cirrhosis in weanling rats following a single small dose of
the Senecio alkaloid, lasiocarpine. Am. J. Pathol. 49, 129−151.
(121) Gordon, G. J., Coleman, W. B., and Grisham, J. W. (2000)
Temporal analysis of hepatocyte differentiation by small hepatocyte-
like progenitor cells during liver regeneration in retrorsine-exposed
rats. Am. J. Pathol. 157, 771−786.
(122) Hanumegowda, U. M., Copple, B. L., Shibuya, M., Malle, E.,
Ganey, P. E., and Roth, R. A. (2003) Basement membrane and matrix
metalloproteinases in monocrotaline-induced liver injury. Toxicol. Sci.
76, 237−246.
(123) Wang, X., Kanel, G. C., and DeLeve, L. D. (2000) Support of
sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive
disease in the rat. Hepatology 31, 428−434.
(124) Ridker, P. M., Ohkuma, S., McDermott, W. V., Trey, C., and
Huxtable, R. J. (1985) Hepatic venooclusive disease associated with
the consumption of pyrrolizidine-containing dietary supplements.
Gastroenterology 88, 1050−1054.
(125) Rasenack, R., Müller, C., Kleinschmidt, M., Rasenack, J., and
Wiedenfeld, H. (2003) Veno-occlusive disease in a fetus caused by
pyrrolizidine alkaloids of food origin. Fetal Diagn. Ther. 18, 223−225.
(126) Kay, J. M., and Heath, D. (1966) Observations on the
pulmonary arteries and heart weight of rats fed on Crotalaria spectabilis
seeds. J. Pathol. Bacteriol. 92, 385−394.
(127) Heath, D., and Kay, J. M. (1967) Medial thickness of
pulmonary trunk in rats with cor pulmonale induced by ingestion of
Crotalaria spectabilis seeds. Cardiovasc. Res. 1, 74−79.
(128) Kay, J. M., Harris, P., and Heath, D. (1967) Pulmonary
hypertension produced in rats by ingestion of Crotalaria spectabilis
seeds. Thorax 22, 176−179.
(129) Burns, J. (1972) The heart and pulmonary arteries in rats fed
on Senecio jacobaea. J. Pathol. 106, 187−194.
(130) Allen, J. R., and Chesney, C. F. (1972) Effect of age on
development of cor pulmonale in non-human primates following
pyrrolizidine alkaloid intoxication. Exp. Mol. Pathol. 17, 220−232.
(131) Chesney, C. F., and Allen, J. R. (1973) Endocardial fibrosis
associated with monocrotaline induced pulmonary hypertension in
16
Perspective
non-human primates (Macaca arctoides). Am. J. Vet. Res. 34, 1577−
1581.
(132) Chesney, C. F., and Allen, J. R. (1973) Monocrotaline induced
pulmonary vascular lesions in non-human primates. Cardiovasc. Res. 7,
508−518.
(133) Fishman, A. P. (1974) Dietary pulmonary hypertension. Circ.
Res. 35, 657−660.
(134) Pan, L. C., Wilson, D. W., Lamé, M. W., Jones, A. D., and
Segall, H. J. (1993) Cor pulmonale is caused by monocrotaline and
dehydromonocrotaline, but not by glutathione or cysteine conjugates
of dihydropyrrolizine. Toxicol. Appl. Pharmacol. 118, 87−97.
(135) Sehgal, P. B., and Mukhopadhyay, S. (2007) Dysfunctional
intracellular trafficking in the pathobiology of pulmonary arterial
hypertension. Am. J. Respir. Cell Mol. Biol. 37, 31−37.
(136) Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., and
McMurtry, I. F. (2009) Animal models of pulmonary arterial
hypertension: the hope for etiological discovery and pharmacological
cure. Am. J. Physiol.: Lung Cell. Mol. Physiol. 297, L1013−1032.
(137) Yen, C. H., Tsai, T. H., Leu, S., Chen, Y. L., Chang, L. T., Chai,
H. T., Chung, S. Y., Chua, S., Tsai, C. Y., Chang, H. W., Ko, S. F., Sun,
C. K., and Yip, H. K. (2013) Sildenafil improves long term effect of
endothelial progenitor cell-based treatment for monocrotaline-induced
rat pulmonary arterial hypertension. Cytotherapy 15, 209−223.
(138) Gupta, V., Gupta, N., Shaik, I. H., Mehvar, R., Nozik-Grayck,
E., McMurtry, I. F., Oka, M., Komatsu, M., and Ahsan, F. (2013)
Inhaled PLGA particles of prostaglandin E1 ameliorate symptoms and
progression of pulmonary hypertension at a reduced dosing frequency.
Mol. Pharmaceutics 10, 1655−1667.
(139) Gomez-Arroyo, J. G., Farkas, L., Alhussaini, A. A., Farkas, D.,
Kraskauskas, D., Voelkel, N. F., and Bogaard, H. J. (2012) The
monocrotaline model of pulmonary hypertension in perspective. Am. J.
Physiol.: Lung Cell. Mol. Physiol. 302, L363−369.
(140) Kay, J. M., Heath, D., Smith, P., Bras, G., and Summerell, J.
(1971) Fulvine and pulmonary circulation. Thorax 26, 249−261.
(141) Heath, D., Shaba, J., Williams, A., Smith, P., and Kombe, A.
(1975) A pulmonary hypertension-producing plant from Tanzania.
Thorax 30, 399−404.
(142) Györika, S., and Stricker, H. (2009) Severe pulmonary
hypertension possibly due to pyrrolizidine alkaloids in polyphytother-
apy. Swiss Med. Wkly. 139, 210−211.
(143) Couet, C. E., Hanley, A. B., MacDonald, S., and Mayes, L.
(1993) The biological assay of natural mutagens using p53, in Food
and Cancer Prevention (Waldron, K. W., Johnston, I. T., and Fenwick,
G. R., Eds.) pp 426−428, Royal Society of Chemistry, London.
(144) Ji, L., Zhang, M., Sheng, Y., and Wang, Z. (2005) Pyrrolizidine
alkaloid clivorine induces apoptosis in human normal liver L-20 cells
and reduces the expression of p53 protein. Toxicol. In Vitro 19, 41−46.
(145) Hirono, I. (1993) Edible plants containing naturally occurring
carcinogens in Japan. Jpn. J. Cancer Res. 84, 997−1006.
(146) Chan, P. C., Haseman, J. K., Prejean, J. D., and Nyska, A.
(2003) Toxicity and carcinogenicity of riddelliine in rats and mice.
Toxicol. Lett. 144, 295−311.
(147) Malkin, D. (2011) Li-Fraumeni syndrome. Genes Cancer 2,
475−484.
(148) LeTendre, L., Ludwig, J., Perrault, J., Smithson, W. A., and
Kovach, J. S. (1984) Hepatocellular toxicity during treatment of
refractory acute leukemia with indicine N-oxide. Cancer 54, 1256−
1259.
(149) Miser, J. S., Smithson, W. A., Krivit, W., Hughes, C., Davis, D.,
Krailo, M., and Hammond, D. (1991) Phase II trial of indicine N-oxide
in relapsed pediatric solid tumors. Invest. New Drugs 9, 339−342.
(150) Rai, P. R., Cool, C. D., King, J. A. C., Stevens, T., Burns, N.,
Winn, R. A., Kasper, M., and Voelkel, N. F. (2008) The cancer
paradigm of severe pulmonary arterial hypertension. Am. J. Respir. Crit.
Care Med. 178, 558−564.
(151) Schoental, R. (1968) Toxicology and carcinogenic action of
pyrrolizidine alkaloids. Cancer Res. 28, 2237−2246.
(152) McLean, E. K. (1970) The toxic actions of pyrrolizidine
(Senecio) alkaloids. Pharmacol. Rev. 22, 429−483.
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
(153) Svoboda, D. J., and Reddy, J. K. (1972) Malignant tumors in
rats given lasiocarpine. Cancer Res. 32, 908−912.
(154) Hirono, I., Shimizu, M., Fushimi, K., Mori, H., and Kato, K.
(1973) Carcinogenic activity of Petasites japonicus Maxim, a kind of
coltsfoot. Gann Monogr. Cancer Res. 64, 527−528.
(155) Hirono, I., Mori, H., and Haga, M. (1978) Carcinogenic
activity of Symphytum of f icinale. J. Natl. Cancer Inst. 61, 865−869.
(156) Kanwar, V. S., Albuquerque, M. L., Ribeiro, R. C., Kauffman,
W. M., and Furman, W. L. (1995) Veno-occlusive disease of the liver
after chemotherapy for rhabdomyosarcoma: case report with a review
of the literature. Med. Pediatr. Oncol. 24, 334−340.
(157) Ortega, J. A., Donaldson, S. S., Ivy, S. P., Pappo, A., and
Maurer, H. M. (1997) Venoocclusive disease of the liver after
chemotherapy with vincristine, actinomycin D, and cyclophosphamide
for the treatment of rhabdomyosarcoma. Cancer 79, 2435−2439.
(158) Bisogno, G., de Kraker, J., Weirich, A., Masiero, L., Ludwig, R.,
Tornade, M. F., and Carli, M. (1997) Veno-occlusive disease of the
liver in children treated for Wilms tumor. Med. Pediatr. Oncol. 29,
245−251.
(159) Ludwig, R., Weirich, A., Abel, U., Hofmann, W., Graf, N., and
Tournade, M. F. (1999) Hepatotoxicity in patients treated according
to the nephroblastoma trial and study SIOP-9/GPOH. Med. Pediatr.
Oncol. 33, 462−469.
(160) Burkhardt, A., and Klöppel, A. (1977) Unusual obliterative
disease of the hepatic veins in an infant. Virchows Arch. A: Pathol. Anat.
Histol. 375, 225−232.
(161) Rubbia-Brandt, L., Audard, V., Sartoretti, P., Roth, A. D.,
Brezault, C., Le Charpentier, M., Dousset, B., Morel, P., Soubrane, O.,
Chausade, S., Mentha, G., and Terris, B. (2004) Severe hepatic
sinusoidal obstruction associated with oxaliplatin-based chemotherapy
in patients with metastatic colorectal cancer. Ann. Oncol. 15, 460−466.
(162) Boula, A. M., Mantadakis, E., Xilouri, I. M., Christoforidou, A.
V., Foudoulakis, A. M., and Samonis, G. (2005) Veno-occlusive disease
of the liver associated with chronic myelomonocytic leukemia treated
with vincristine and standard doses of cytarabine. Am. J. Hematol. 79,
216−219.
(163) Elli, M., Pinarli, F. G., Dagdemir, A., and Acar, S. (2006) Veno-
occlusive disease of the liver in a child after chemotherapy for brain
tumor. Pediatr. Blood Cancer 46, 521−523.
(164) Matute-Bello, G., McDonald, G. D., Hinds, M. S., Schoch, H.
G., and Crawford, S. W. (1998) Association of pulmonary function
testing abnormalities and severe veno-occlusive disease of the liver
after marrow transplantation. Bone Marrow Transplant. 21, 1152−
1130.
(165) McDonald, G. B., Hinds, M. S., Fisher, L. D., Schoch, H. G.,
Wolford, J. L., Banaji, M., Hardin, B. J., Shulman, H. M., and Clift, R.
A. (1993) Veno-occlusive disease of the liver and multiorgan failure
after bone marrow transplantation: a cohort study of 355 patients. Ann.
Int. Med. 118, 255−267.
(166) Strasser, S. I., Sullivan, K. M., Myerson, D., Spurgeon, C. L.,
Storer, B., Schoch, H. G., Murakami, C. S., and McDonald, G. B.
(1999) Cirrhosis of the liver in long-term marrow transplant survivors.
Blood 93, 3259−3266.
(167) Copelan, E. A., Penza, S. L., Elder, P. J., Ezzone, S. A., Scholl,
M. D., Bechtel, T. P., Belt, P. S., and Avalos, B. R. (2000) Analysis of
prognostic factors for allogenic marrow transplantation following
busulfan and cyclophosphamide in myelodysplastic syndrome and
after leukemic transformation. Bone Marrow Transplant. 25, 1219−
1222.
(168) Horn, B., Reiss, U., Matthay, K., McMillan, A., and Cowan, M.
(2002) Veno-occlusive disease of the liver in children with solid
tumors undergoing autologous hematopoietic progentitor cell trans-
plantation: a high incidence in patients with neuroblastoma. Bone
Marrow Transplant. 29, 409−415.
(169) Bairey, O., Kirgner, I., Yakobi, M., Hamdan, A., Ben-Ari, Z.,
and Shaklai, M. (2002) Clinical severe hepatic venooclusive disease
during induction treatment of acute monoblastic leukemia managed
with defibrotide. Am. J. Hematol. 69, 281−284.
17
Perspective
(170) Barker, C. C., Butzner, J. D., Anderson, R. A., Brant, R., and
Sauve, R. S. (2003) Incidence, survival and risk factors for the
development of veno-occlusive disease in pediatric hematopoietic stem
cell transplant recipients. Bone Marrow Transplant. 32, 79−87.
(171) Kraemer, D. M., Waschke, J., and Kunzmann, V. (2003)
Venoocclusive disease in a male patient with Marfam syndrome and
common acute lymphoblastic leukemia during induction therapy. Ann.
Hematol. 82, 444−447.
(172) Bajwa, R. P. S., Cant, A. J., Abinun, M., Flood, T. J., Hodges, S.,
Hale, J. P., and Skinner, R. (2003) Recombinant tissue plasminogen
activator for treatment of hepatic veno-occlusive disease following
bone marrow transplantation in children: effectiveness and a scoring
system for initiating treatment. Bone Marrow Transplant. 31, 591−597.
(173) Kalayoglu-Besisik, S., Yenerel, M. N., Caliskan, Y., Ozturk, S.,
Besisik, F., and Sargin, D. (2005) Time-related changes in the
incidence severity and clinical outcome of hepatic veno-occlusive
disease in hematopoietic stem cell transplantation patients during the
past ten years. Transplant. Proc. 37, 2285−2289.
(174) Papadopoulos, A., Ntaios, G., Kaiafa, G., Girtovitis, Z. S.,
Kontoninas, Z., Diamantidis, M. D., Savopoulos, C., and Hatzitolios, A.
(2008) Veno-occlusive disease of the liver during induction therapy for
acute lymphoblastic leukemia. Int. J. Hematol. 88, 441−442.
(175) Dulley, F. L., Kanfer, E. J., Appelbaum, F. R., Amos, D., Hill, R.
S., Buckner, C. D., Shulman, H. M., McDonald, G. B., and Thomas, E.
D. (1987) Veno-occlusive disease of the liver after chemoradiotherapy
and autologous bone marrow transplantation. Transplantation 43,
870−873.
(176) Carreras, E. (2000) Veno-occlusive disease of the liver after
hemopoietic cell transplantation. Eur. J. Haematol. 64, 281−291.
(177) Kumar, S., DeLeve, L. D., Kamath, P. S., and Tefferi, A. (2003)
Hepatic veno-occlusive disease (sinusoidal obstruction syndrome)
after hematopoietic stem cell transplantation. Mayo Clin. Proc. 78,
589−598.
(178) Gharib, M. I., Bulley, S. R., Doyle, J. J., and Wynn, R. F. (2006)
Venous occlusive disease in children. Thromb. Res. 118, 27−38.
(179) Rubbia-Brandt, L., Tauzin, S., Brezault, C., Delucinge-Vivier,
C., Descombes, P., Dousset, P. E., Mentha, G., and Terris, B. (2011)
Gene expression profiling provides insights into pathways of
oxaliplatin-related sinusoidal obstruction syndrome in humans. Mol.
Cancer Ther. 10, 687−696.
(180) De Jonge, M. E., Huitema, A. D. R., Beijnen, J. H., and
Rodenhuis, S. (2006) High exposures to bioactivated cyclophospha-
mide are related to the occurrence of veno-occlusive disease of the
liver following high-dose chemotherapy. Br. J. Cancer 94, 1226−1230.
(181) Plapp, F. V., and Chiga, M. (1972) Disaggregation of mouse
liver polysomes by cyclophosphamide. Arch. Pharmacol 272, 351−353.
(182) Yan, C. C., and Huxtable, R. J. (1996) Effects of
monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in
the rat. Biochem. Pharmacol. 51, 375−379.
(183) Hettmer, S., and Wagers, A. J. (2010) Uncovering the origins
of rhabdomyosarcoma. Nat. Med. 16, 171−173.
(184) Daughton, C. G. (2005) Overlooked in Fallon? Environ. Health
Perspect. 113, A224.
(185) Morty, R. E., Nejman, B., Kwapiszewska, G., Hecker, M.,
Zakrzewicz, A., Kouri, F. M., Peters, D. M., Dumitrascu, R., Seeger, W.,
Knaus, P., Schermuly, R. T., and Eickelberg, O. (2007) Dysregulated
bone morphogenetic Protein signaling in monocrotaline-induced
pulmonary arterial hypertension. Arterioscler., Thromb., Vasc. Biol. 27,
1072−1078.
(186) Murakami, K., Mathew, R., Huang, J., Farahani, R., Peng, H.,
Olson, S. C., and Etlinger, J. D. (2010) Smurf1 ubiquitin ligase causes
downregulation of BMP receptors and is induced in monocrotaline
and hypoxia models of pulmonary arterial hypertension. Exp. Biol.
Med. 235, 805−813.
(187) Ramos, M., Lamé, M. W., Segall, H. J., and Wilson, D. W.
(2007) Monocrotaline pyrrole induces Smad nuclear accumulation
and altered signaling expression in human pulmonary arterial
endothelial cells. Vasc. Pharmacol. 46, 439−448.
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
(188) Owens, G. K., Rabinovitch, P. S., and Schwartz, S. M. (1981)
Smooth muscle hypertrophy versus hyperplasia in hypertension. Proc.
Natl. Acad. Sci. U.S.A. 78, 7759−7763.
(189) Veyssier-Belot, C., and Cacoub, P. (1999) Role of endothelial
and smooth muscle cells in the physiopathology and treatment
management of pulmonary hypertension. Cardiovasc. Res. 44, 274−
282.
(190) Tajsic, T., and Morrell, N. W. (2011) Smooth muscle cell
hypertrophy, proliferation, migration and apoptosis in pulmonary
hypertension. Comp. Physiol. 1, 295−317.
(191) Lappin, P. B., and Roth, R. A. (1997) Hypertrophy and
prolonged DNA synthesis in smooth muscle cells characterize
pulmonary arterial wall thickening after monocrotaline pyrrole
administration. Toxicol. Pathol. 25, 372−380.
(192) Lappin, P. B., Ross, K. L., King, L. E., Fraker, P. J., and Roth, R.
A. (1998) The response of pulmonary vascular endothelial cells to
monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro
and in vivo. Toxicol. Appl. Pharmacol. 150, 37−48.
(193) Lee, J., Reich, R., Xu, F., and Sehgal, P. B. (2009) Golgi,
trafficking, and mitosis dysfunction in pulmonary arterial endothelial
cells exposed to monocrotaline pyrrole and NO scavenging. Am. J.
Physiol.: Lung Cell. Mol. Physiol. 297, L715−L728.
(194) Mukhopadhyay, S., Shah, M., Xu, F., Patel, K., Tuder, R. M.,
and Sehgal, P. B. (2008) Cytoplasmic provenance of STAT3 and PY-
STAT3 in the endolysomal compartments in pulmonary arterial
endothelial and smooth muscle cells: implications in pulmonary
arterial hypertension. Am. J. Physiol.: Lung Cell Mol. Physiol. 249,
L449−L468.
(195) Bull, L. B. (1955) The histological evidence of liver damage
from pyrrolizidine alkaloids: megalocytosis of the liver cells and
inclusion globules. Aust. Vet. J. 31, 33−40.
(196) Jago, M. V. (1969) The development of the hepatic
megalocytosis of chronic pyrrolizidine alkaloid poisoning. Am. J.
Pathol. 56, 405−422.
(197) Svoboda, D., Reddy, J., and Bunyaratvej, S. (1971) Hepatic
megalocytosis in chronic lasiocarpine poisoning. Am. J. Pathol. 65,
399−408.
(198) Samuel, A., and Jago, M. V. (1975) Localization in the cell
cycle of the antimitotic action of the pyrrolizidine alkaloid lasiocarpine
and of its metabolite, dehydroheliotridine. Chem.−Biol. Interact. 10,
185−197.
(199) Runo, J. R., and Loyd, J. E. (2003) Primary pulmonary
hypertension. Lancet 361, 1533−1544.
(200) Aldred, M. A., Comhair, S. A., Varella-Garcia, M., Asosingh, K.,
Xu, W., Noon, G. P., Thistlewaite, P. A., Tuder, R. M., Erzurum, S. C.,
Geraci, M. W., and Coldren, C. D. (2010) Somatic chromosome
abnormalities in the lungs of patients with pulmonary arterial
hypertension. Am. J. Respir. Crit. Care Med. 182, 1153−1160.
(201) Austin, E. D., Hamid, R., and Ahmed, F. (2010) Somatic
mutations in pulmonary arterial hypertension. Am. J. Respir. Crit. Care
Med. 182, 1095−1095.
(202) Taura, P., Garcia-Valdecasas, J. C., Beltran, J., Izquierdo, E.,
Navasa, M., Sala-Blanch, J., Mas, A., Balust, J., Grande, L., and Visa, J.
(1996) Moderate primary pulmonary hypertension in patients
undergoing liver transplantation. Anesth. Analg. 83, 675−680.
(203) Hoeper, M. M., Krowka, M. J., and Strassburg, C. P. (2004)
Portopulmonary hypertension and hepatopulmonary syndrome.
Lancet 363, 1461−1468.
(204) Herve, P., Le Pavec, J., Sztrymf, B., Decante, B., Savale, L., and
Sitbon, O. (2007) Pulmonary vascular abnormalities in cirrhosis. Best
Pract. Res., Clin. Gastroenterol. 21, 141−159.
(205) Kochar, R., Moises, I., Rubin, N., and Fallon, M. B. (2011)
Pulmonary complications of cirrhosis. Curr. Gastroenterol. Rep. 13, 34−
39.
(206) McDonnell, P. J., Toye, P. A., and Hutchins, G. M. (1983)
Primary pulmonary hypertension and cirrhosis: are they related? Am.
Rev. Respir. Dis. 127, 437−441.
18
Perspective
(207) Murata, K., Shimizo, A., Takase, K., Nakano, T., and Tameda,
Y. (1997) Asymptomatic primary pulmonary hypertension associated
with liver cirrhosis. J. Gastroenterol. 32, 102−104.
(208) Bragdon, B., Moseychuk, O., Saldanha, S., King, D., Julian, J.,
and Nohe, A. (2011) Bone morphogenetic proteins: a critical review.
Cell. Signalling 23, 609−620.
(209) Okamoto, M., Murai, J., Yoshikawa, H., and Tsumali, N.
(2006) Bone morphogenetic proteins in bone stimulate osteoclasts
and osteoblasts during bone development. J. Bone Miner. Res. 21,
1022−1033.
(210) Steward, C. G., Pellier, I., Mahajan, A., Ashworth, M. T., Stuart,
A. G., Fasth, A., Lang, D., Fischer, A., Friedrich, W., and Schulz, A. S.
(2004) Severe pulmonary hypertension: a frequent complication of
stem cell transplantation for malignant infantile osteopetrosis. Br. J.
Hamaetol. 124, 63−71.
(211) Slatter, M. A., Rao, K., Amrolia, P., Flood, T., Abinun, M.,
Hambleton, S., Nademi, Z., Goulden, N., Davies, G., Qasim, W.,
Gaspar, H. B., Cant, A., Gennery, A. R., and Veys, P. (2011)
Treosulfan-based conditioning regimens for hematopoietic stem cell
transplantation in children with primary immunodeficiency (PID): UK
experience. Blood 117, 4367−4375.
(212) Kaplan, F. S., Zasloff, M. A., Kitterman, J. A., Shore, E. M.,
Hong, C. X., and Rocke, D. M. (2010) Early mortality and
cardiorespiratory failure in patients with fibrodysplasia ossificans
progressiva. J. Bone Jt. Surg., Am. Vol. 92, 686−691.
(213) Xu, M.-Q., Feldman, G., LeMerrer, M., Shugart, Y. Y., Glaser,
D. L., Urtizberea, J. A., Fardeau, M., Connor, J. M., Triffitt, J., Smith,
R., Shore, E. M., and Kaplan, F. S. (2000) Linkage exclusion and
mutational analysis of the noggin gene in patients with fibrodysplasia
ossificans progressive (FOP). Clin. Genet. 58, 291−298.
(214) Gannon, F. H., Kaplan, F. S., Olmsted, E., Finkel, G. C.,
Zasloff, M. A., and Shore, E. S. (1997) Bone morphogenetic protein 2/
4 in early fibromatous lesions of fibrodysplasia ossificans progressive.
Human Pathol. 28, 339−343.
(215) Enklaar, T., Zabel, B. U., and Prawitt, D. (2006) Beckwith-
Wiedemann syndrome: multiple molecular mechanisms. Expert Rev.
Mol. Med. 8, 1−19.
(216) Sun, J., Liu, Y.-H., Chen, H., Nguyen, M. P., Mishina, Y.,
Upperman, J. S., Ford, H. R., and Shi, W. (2007) Deficient Alk3-
mediated BMP signaling causes prenatal omphalocele-like defect.
Biochem. Biophys. Res. Commun. 360, 238−243.
(217) Sotelo-Avila, C., and Gooch, W. M. (1976) Neoplasms
associated with the Beckwith-Weidemann syndrome. Perspect. Pediatr.
Pathol. 3, 255−272.
(218) Rump, P., Zeegers, M. P. A., and van Essen, A. J. (2005)
Tumor risk in Beckwith-Wiedemann syndrome: a review and meta-
analysis. Am. J. Med. Gen. 136A, 95−104.
(219) Spector, L. G., and Birch, J. (2012) The epidemiology of
hepatoblastoma. Pediatr. Blood Cancer 59, 776−779.
(220) Bardeesy, N., Falkoff, D., Petruzzi, M., Nowak, N., Zabel, B.,
Adam, M., Agiar, M. C., Grundy, P., Shows, T., and Pelletier, J. (1994)
Anaplastic Wilm’s tumour, a subtype displaying poor prognosis,
harbours p53 gene mutations. Nat. Genet. 7, 91−97.
(221) Muwakkit, S., Antillon, F., Valverde, P., Jenkins, J. J., Zaatari,
G., and Dome, J. S. (2002) Letter to the Editor: Simultaneous
occurrence of Wilms tumor and rhabdomyosarcoma in two patients.
Med. Pediatr. Oncol. 39, 143−145.
(222) Müller-Höcker, J., Weiss, M., Meyer, U., Schramel, P.,
Wiebecke, B., Belohradsky, B. H., and Hübner, G. (1987) Fatal
copper storage disease of the liver in a German infant resembling
Indian childhood cirrhosis. Virchows Arch. A: Pathol. Anat. Histopathol.
411, 379−385.
(223) Price, L. A., Walker, N. I., Clague, A. E., Pullen, I. D., Smits, S.
J., Ong, T.-H., and Patrick, M. (1996) Chronic copper toxicosis
presenting as liver failure in an Australian child. Pathology 28, 316−
320.
(224) Seibold-Weiger, K., Vochem, M., Mackensen-Haen, S., and
Speer, C. (1997) Fatal hepatic veno-occlusive disease in a newborn
infant. Am. J. Perinatol. 14, 107−111.
DOI: 10.1021/tx500403t
Chem. Res. Toxicol. 2015, 28, 4−20
Chemical Research in Toxicology
(225) Sergi, C., Beedgen, B., Linderkamp, O., and Hofmann, W. J.
(1999) Fatal course of veno-occlusive disease of the liver
(Endophlebitis Hepatica obliterans) in a preterm infant. Pathol., Res.
Pract. 195, 847−851.
(226) Trollmann, R., Neureiter, D., Lang, T., Dörr, H. G., and
Behrens, R. (1999) Late manifestation of Indian childhood cirrhosis in
a 3-year-old German girl. Eur. J. Pediatr. 158, 375−378.
(227) Bull, L. B., Dick, A. T., Keast, J. C., and Edgar, G. (1956) An
experimental investigation of the hepatotoxic and other effects on
sheep of consumption of Heliotropium europaeum L.: heliotrope
poisoning of sheep. Aust. J. Agric. Res. 7, 281−331.
(228) St George-Grambauer, T. D., and Rac, R. (1962)
Hepatogenous chronic copper poisoning in sheep in South Australia
due to the consumption of Echium plantagineum L. (Salvation Jane).
Aust. Vet. J. 38, 288−293.
(229) Miranda, C. L., Henderson, M. C., and Buhler, D. R. (1981)
Dietary copper enhances the hepatotoxicity of Senecio jacobaea in rats.
Toxicol. Appl. Pharmacol. 60, 418−423.
(230) Howell, J. M., Deol, H. S., Dorling, P. R., and Thomas, J. B.
(1991) Experimental copper and heliotrope intoxication in sheep:
morphological changes. J. Comp. Pathol. 105, 49−74.
(231) Walker-Smith, J., and Blomfield, J. (1973) Wilson’s disease or
chronic copper poisoning? Arch. Dis. Child. 48, 476−479.
(232) Müller-Höcker, J., Meyer, U., Wiebecke, B., Hübner, G., Eife,
R., Kellner, M., and Schramel, P. (1988) Copper storage disease of the
liver and chronic dietary copper intoxication in two further German
infants mimicking Indian Childhood Cirrhosis. Pathol., Res. Pract. 183,
39−45.
(233) Müller-Höcker, J., Summer, K. H., Schramel, P., and Rodeck,
B. (1998) Different pathomorphic patterns in exogenic infantile
copper intoxication of the liver. Pathol., Res. Pract. 194, 377−384.
(234) Von Mühlendahl, K. E., and Lange, H. (1994) Copper and
childhood cirrhosis. Lancet 344, 1515−1516.
(235) Scheinberg, I. H., and Sternlieb, I. (1994) Is non-Indian
childhood cirrhosis caused by excess dietary copper? Lancet 344,
1002−1004.
(236) Baker, A., Gormally, S., Saxena, R., Baldwin, D., Drumm, B.,
Bonham, J., Potmann, B., and Mowat, A. P. (1995) Copper-associated
liver disease in childhood. J. Hepatol. 23, 538−543.
(237) Müller, T., Feichtinger, H., Berger, H., and Müller, W. (1996)
Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder. Lancet
347, 877−880.
(238) Müller, T., Müller, W., and Feichtinger, H. (1998) Idiopathic
copper toxicosis. Am. J. Clin. Nutr. 67, 1082S−1986S.
(239) Wijmenga, C., Müller, T., Brunt, T., Feichtinger, H.,
Schönitzer, D., Houwen, R. H. J., Müller, W., Sandkuijl, L. A., and
Pearson, P. L. (1998) Endemic Tyrolean infantile cirrhosis is not an
allelic variant of Wilson’s disease. Eur. J. Hum. Genet. 6, 624−628.
(240) Tanner, M. S. (1998) Role of copper in Indian childhood
cirrhosis. Am. J. Clin. Nutr. 67, 1074S−1081S.
(241) Aston, N. S., Morris, P. A., Tanner, M. S., and Variend, S.
(1998) An animal model for copper-associated cirrhosis in infancy. J.
Pathol. 186, 215−221.
(242) Dieter, H. H., Schimmelpfennig, W., Meyer, E., and Tabert, M.
(1999) Early childhood cirrhosis (ECC) in Germany between 1982
and 1994 with special consideration of copper etiology. Eur. J. Med.
Res. 28, 233−242.
(243) Zietz, B. P., de Vergara, J. D., and Dunkelberg, H. (2003)
Copper concentrations in tap water and possible effects on infant’s
healthresults of a study in Lower Saxony, Germany. Environ. Res. 92,
129−138.
(244) Zietz, B. P., Dieter, H. H., Lakomek, M., Schneider, H.,
Kessler-Gaedke, B., and Dunkelberg, H. (2003) Epidemiological
investigation on chronic copper toxicity to children exposed via the
public drinking water supply. Sci. Total Environ. 302, 127−144.
(245) Mellis, C., and Bales, P. M. (1976) Familial hepatic
venoocclusive disease with probable immune deficiency. J. Pediatr.
88, 236−242.
19
Perspective
(246) Etzioni, A., Benderly, A., Rosenthal, E., Shehadah, V.,
Auslander, L., Lahat, N., and Pollack, S. (1987) Defective humoral
and cellular immune functions associated with veno-occlusive disease
of the liver. J. Pediatr. 110, 549−554.
(247) Roscioli, T., Cliffe, S. T., Bloch, D. B., Bell, C. G., Mullan, G.,
Taylor, P. J., Sarris, M., Wang, J., Donald, J. A., Kirk, E. P., Ziegler, J. B.,
Salzer, U., McDonald, G. B., Wong, M., Lindeman, R., and Buckley, M.
F. (2006) Mutations in the gene encoding the PML nuclear body
protein Sp110 are associated with immunodeficiency and hepatic
veno-occlusive disease. Nat. Genet. 38, 620−622.
(248) Cliffe, S. T., Wong, M., Taylor, P. J., Ruga, E., Wilcken, B.,
Lindeman, R., Buckley, M. F., and Roscioli, T. (2007) The first
prenatal diagnosis for veno-occlusive disease and immunodeficiency
syndrome, an autosomal recessive condition associated with mutation
in SP110. Prenatal Diagn. 27, 674−676.
(249) Cliffe, S. T., Bloch, D. B., Suryani, S., Kamsteeg, E.-J., Avery, D.
T., Palendira, U., Church, J. A., Wainstein, B. K., Trizzino, A., Lefranc,
G., Akatcherian, C., Megarbane, A., Gilissen, C., Moshous, D.,
Reichenbach, J., Ziegler, J. B., Wong, M., Tangye, S. G., Lindeman,
R., Buckley, M. F., and Roscioli, T. (2012) Clinical, molecular, and
cellular immunological findings in patients with SP110-associated
veno-occlusive disease with immunodeficiency syndrome. J. Allergy
Clin. Immunol. 130, 735−742.
(250) Wang, T., Ong, P., Roscioli, T., Cliffe, S. T., and Church, J. A.
(2012) Hepatic veno-occlusive disease with immunodeficiency
(VODI): first reported case in the U.S. and identification of a unique
mutation in Sp110. Clin. Immunol. 145, 102−107.
(251) Ganaiem, H., Eisenstein, E. M., Tenenbaum, A., Somech, R.,
Simanovsky, N., Roscioli, T., Weintraub, M., and Stepensky, P. (2013)
The role of hematopoietic stem cell transplantation in SP110
associated veno-occlusive disease with immunodeficiency syndrome.
Pediatr. Allergy Immunol. 24, 250−256.
(252) (2009) Genetics Home Reference, US National Library of
Medicine, http://ghr.nlm.nih.gov/gene/SP110.
(253) Deyo, J. A., and Kerkvliet, N. I. (1990) Immunotoxicity of the
pyrrolizidine alkaloid monocrotaline following subchronic adminis-
tration to C57B1/6 mice. Fundam. Appl. Toxicol. 14, 842−849.
(254) Molyneux, R. J., Gardner, D. L., Colegate, S. M., and Edgar, J.
A. (2011) Pyrrolizidine alkaloid toxicity in livestock: a paradigm for
human poisoning? Food Addit. Contam., Part A 28, 293−307.
(255) Newberne, P. M., and Rogers, A. E. (1973) Nutrition,
monocrotaline and aflatoxin B1 in liver carcinogenesis. Plant Foods
Man 1, 23−31.
(256) Yee, S. B., Kinser, S., Hill, D. A., Barton, C. C., Hotchkis, J. A.,
Harkema, J. R., Ganey, P. E., and Roth, R. A. (2000) Synergistic
hepatotoxicity from coexposure to bacterial endotoxin and the
pyrrolizidine alkaloid monocrotaline. Toxicol. Appl. Pharmacol. 166,
173−185.
(257) (1992) Abwehr von Arzneimittelrisiken-Stufe II. Bundesan-
zeiger 17 June, 4805; cited by Dtsch. Apoth. Ztg. 132, 1406−1408.
(258) European Medicines Agency (2013) Public statement on the use
of herbal medicinal products containing toxic, unsaturated pyrrolizidine
alkaloids (PAs), EMA/HMPC/893108/2011, 2nd draft, 6 Nov,
EMA,London, http://www.ema.europa.eu/docs/en_GB/document_
library/Public_statement/2013/11/WC500154224.pdf.
(259) Czauderna, P., Katski, K., Kowalczyk, J., Kurylak, A., Lopatka,
B., Skotnicka-Klonowicz, G., Sawicz-Birkoska, K., and Godzinski, J.
(2000) Venoocclusive liver disease (VOD) as a complication of
Wilms’ tumour management in the series of consecutive 206 patients.
Eur. J. Pediatr. Surg. 10, 300−303.
(260) Cecen, E., Uysal, K. M., Ozguven, A., and Gunes, D. (2007)
Veno-occlusive disease in a child with rhabdomyosarcoma after
conventional chemotherapy. Pediatr. Hematol. Oncol. 24, 615−621.
(261) Xia, Q., Zhao, Y., Von Tungeln, L. S., Doerge, D. R., Lin, G.,
Cai, L., and Fu, P. P. (2013) Pyrrolizidine alkaloid-derived DNA
adducts as a common biological biomarker of pyrrolizidine alkaloid-
induced tumorigenicity. Chem. Res. Toxicol. 26, 1384−1396.
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