Pyrrolizidine alkaloids in human diet

15 Pages·1999·0.24 MB·English

MutationResearch443(cid:14)1999.53–67
www.elsevier.comrlocatergentox
Communityaddress:www.elsevier.comrlocatermutres
Pyrrolizidine alkaloids in human diet
Arungundrum S. Prakash a,), Tamara N. Pereira a, Paul E.B. Reilly b,
Alan A. Seawright a
a NationalResearchCentreforEn˝ironmentalToxicology,39,KesselsRoad,CoopersPlains,QLD4108,Australia
bDepartmentofBiochemistry,TheUni˝ersityofQueensland,St.Lucia,QLD4072,Australia
Received7October1998;receivedinrevisedform3December1998;accepted10December1998
Abstract
Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion,
metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with
cellularmacromoleculesformingadductswhichmayinitiateacuteorchronictoxicity.Thepyrrolizidinealkaloidspresenta
serious health risk to human populations that may be exposed to them through contaminationof foodstuffs or when plants
containingthemareconsumedasmedicinalherbs.Somepyrrolizidinealkaloids(cid:14)PA.adductsarepersistentinanimaltissue
andthemetabolitesmaybere-releasedandcausedamagelongaftertheinitialperiodofingestion.PAsarealsoknowntoact
as teratogensand abortifacients. Chronic ingestionof plants containingPAs has also led to cancer in experimentalanimals
and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimuriumrmammalianmicrosome
system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the
extensivereportsontheoutcomeofhumanexposureavailableintheliterature,weconcludethatwhilehumansfacetherisk
of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans. q1999 Elsevier Science B.V. All
rightsreserved.
Keywords:Pyrrolizidinealkaloid;Veno-occlusivedisease;Pyrrole;Cancer;Megalocytosis;Antimitoticeffect
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1. Introduction PAs 1. The main sources of toxic alkaloids are the
families—Boraginaceae (cid:14)all genera., Compositae
1.1. Plants containing pyrrolizidine alkaloids (cid:14)tribes Senecionae and Eupatoriae. and Legumi-
nosae (cid:14)genus Crotalaria. w2x. Within these families,
Pyrrolizidine alkaloids (cid:14)PAs. are found in plants
toxic alkaloids are found mainly in the Senecio
of widespread geographical distribution. Over 200
(cid:14)Compositae., Crotalaria (cid:14)Leguminosae. and He-
alkaloidshavebeenidentifiedin300plantspeciesof
w x liotropium (cid:14)Boraginaceae. species.
upto13families 1,26,75.Ithasbeenestimatedthat
PAs are esters of hydroxylated 1-methylpyrro-
up to 3% of the world flowering plants contain toxic
lizidines. Hepatotoxic PAs are esters of unsaturated
necines having a 1,2 double bond. The structure ofPAs is composed of necine, two fused five-mem-
)Corresponding author. Tel.: q61-7-32749002; Fax: q61-7- bered rings joined by a single nitrogen atom, and
32749003;E-mail:[email protected]
1383-5718r99r$-seefrontmatterq1999ElsevierScienceB.V.Allrightsreserved.
PII: S1383-5742(cid:14)99.00010-1
54 A.S.Prakashetal.rMutationResearch443(1999)53–67
thatmuchofthePAswereexcretedintheskim-milk
fractionanddeducedthatthesemaybewater-soluble
metabolites. Later, water-soluble PA N-oxides were
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identified in milk 9. Milk from lactating rats fed
PAs were shown to be mutagenic in the
Salmonellarmammalian-microsome mutagenicity
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test 10 and in the Drosophilla sex-linked recessive
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lethal assay 11. It was also demonstrated that liver
lesions (cid:14)centrilobular megalocytes and biliary ductu-
lar hyperplasia. could be elicited in rats consuming
milkfromlactatinggoatsfed Seneciojacobaea(cid:14)tansy
ragwort. w9x. Similar lesions were seen when the rats
were directly fed a diet containing 0.001–1% S.
jacobaea.
2.3. PAs in medicinal plants
PAs have been identified in traditional herbal
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medicines of South America 12,13, Sri-Lanka
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14,15 and China 16,17. Of the herbal remedies
containing pyrrolizidine alkaloids, comfrey has re-
Fig.1.Representativepyrrolizidinealkaloidstructures. ceived the most attention. Studies have shown the
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presence of toxic PAs in fresh leaves 18, commer-
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cialcomfreypreparations 19 andincomfrey-pepsin
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capsules 20. Following an episode of comfrey-
necic acid which is made up of one or two car- pepsin related poisoning w21x the sale of comfrey
boxylic ester arms which may form a macrocyclic products for internal use was banned in the US and
structure. Fig. 1 shows the structures of some com- in Canada w22x. Nevertheless, comfrey leaves and
monly known PAs (cid:14)Fig. 1.. extracts continue to be used in poultices, creams and
ointmentsfortopicalapplication.Comfreyleavesare
consumed in salads, particularly in Europe, North
2. Pathways of human exposure America, Japan and Australia. Toxic PAs have also
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been isolated from leaves of H. digyum 23, which
2.1. Contamination of staple food are consumed in East Africa.
Large outbreaks of poisoning have occurred 2.4. Honey
throughcontaminationofwheatcropsinAfghanistan,
India and the former USSR w3–5x. This is made The presence of the alkaloids (cid:14)seneciphyline,
possiblebecausethePAcontainingplants,inparticu- senecionine, jacobine, jaconine, jacoline and jacoz-
lar, three species of Boraginaceae, Heliotropium la- ine. in honey produced by bees foraging in a region
siocarpum, H. popo˝ii and H. europaeum, are well of Oregon infested with S. jacobaea (cid:14)tansy ragwort.
adaptedtovigorousgrowthundertheclimaticcondi- has been reported w24x. The honey also contained
tions in which wheat is usually grown. small amounts of ragwort pollen. The PA content
was estimated to be at 0.3–3.9 ppm. Similarly, Cul-
2.2. Milk venor et al. w25x found alkaloids (cid:14)echimidine and
smaller amounts of 7-acetyllycopsamine, 7-acetylin-
LaboratorystudieshaveshownPAsarepresentin termedine, echiumine, uplandicine, lycopsamine, in-
milkfromcowsw6xandgoatsw7x.Eastmanw8xshowed termedine and acetylechimidine. at 0.54–1.9 ppm in
A.S.Prakashetal.rMutationResearch443(1999)53–67 55
3. Structural features of PAs
3.1. Features for hepatotoxicity
A comprehensive account of the various naturally
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occurring PA structures are given by Mattocks 26.
The potential of PA compounds as hepatotoxins is
governedby certain minimumstructuralfeatures: (cid:14)1.
an unsaturated 3-pyrroline ring, (cid:14)2. one or two hy-
droxylgroupseachattachedtothepyrrolinering,(cid:14)3.
one or preferably two esterified groups and (cid:14)4. the
presence of a branched chain on the acid moiety
Fig.2. StructuralfeaturesessentialforPAtoxicity.Thenumbers
correspondtotheitemslistedinSection3. (cid:14)Fig. 2..
3.2. Steps in the production of toxic metabolite
honey from regions of southeastern Australia where
bees forage on Echium plantagineum (cid:14)Patterson’s A schematic representation of the PA metabolic
Curse or Salvation Jane.. pathway is shown in Fig. 3. This diagram is a
Fig.3.Themetabolicpathwayleadingtotoxicityofpyrrolizidinealkaloids.
56 A.S.Prakashetal.rMutationResearch443(1999)53–67
modified version of the one published by Roeder appears to be predominantly carried out in the liver
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27. Essentially, the parent PA is either hydrolysed but the particular esterase isoforms responsible ap-
to non-toxic necines and necic acids or to ester pear not to have been extensivelycharacterised. This
pyrroles by esterases or P450 enzymes respectively. metabolic route is very important after exposure to
Theesterpyrroles(cid:14)EPy.areconsideredtobehepato- these compounds because esterase cleavage is a
toxic due to their high reactivity while the less detoxication pathway, promoting the clearance of
reactive longer-lived alcoholic pyrroles (cid:14)APy., pro- these xenobiotics as non-toxic products. Esterase
duced by the hydrolysis of the EPy, lead to antimi- activitytowardsmonocrotalineis particularlyhighin
totic effects and to mutagenic and carcinogenic le- guinea pig liver w30x and this is regarded as one
sions. reason for the marked resistance to the toxic effects
of this and mostotherpyrrolizidinealkaloidscharac-
teristic of this species. One exception is jacobine
4. Metabolism of pyrrolizidine alkaloids which is toxic to guinea pigs, and guinea pig liver
microsomesandpurifiedlivercarboxylesteraseshave
There are some hundreds of naturally occurring been shown not to be active in the hydrolysis of
pyrrolizidine alkaloids but most of what is known jacobine w31x. Rat liver microsomes have zero es-
about their metabolism and the molecular basis of terase activity towards monocrotaline and rats are
their toxic effects comes from studies with a limited accordingly susceptible to the toxic actions of this
number of representative compounds with most compound w30x. Esterase activity of human tissues
studies carried out using just a few species of expe- directed towards pyrrolizidine alkaloids appears not
rimental animal. Monocrotaline, senecionine, to have been assessed.
seniciphylline, jacobine, lasiocarpine, ridelliine and A second route of metabolism of these alkaloids
heliotrine appear to have been the most frequently is formation of the N-oxide derivative by microso-
studied alkaloids and rats, pigs, rabbits and guinea mal monooxygenases. N-oxide formation is another
pigs have been used most often to study the detoxication route whose importance varies widely
metabolism and molecular toxicity of these com- between species and it appears that differential sub-
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pounds 28,29. Onlya smallnumberof studieshave strate selectivities of multiple enzymes in different
used human organ donor tissue samples or prepara- organs are responsible for this variation. The mono-
tions of human tissue enzymes to characterise these oxygenases generally found to be most important in
biotransformations as they pertain specifically to thisbiotransformationarethelivermicrosomalflavin
people. monooxygenases. In pig liver for example these
Theparentalkaloidischemicallyunreactive.Once enzymes are very active in the N-oxidation of
ingested, much of it is excreted unchanged but the senecionine w32x but the corresponding enzymes do
remainder is metabolised in the liver. Activation not contribute greatly to this biotransformationin rat
requiresthedehydrogenationofPAstopyrroles(cid:14)Fig. liver w33x. Purified flavin monooxygenase from rab-
3.whichareelectrophilicandwillreactwithnucleo- bit lung was also shown to be inactive in this oxida-
philic tissue components such as nucleic acids and tion reaction w32x. By contrast the flavin mono-
proteins. Since the liver is the site of toxic pyrrole oxygenases of guinea pig lung, liver and kidney
production, it is one of the two main target organs, microsomeswerepresumptivelyidentifiedtobevery
the lungs being the other. activeincatalysingsenecionineN-oxidationw34x and
There are three principal routes by which these this is recognised as another important factor that
compounds are metabolised, with liver being the explains this species’ resistance to the toxic effects
predominantorganinwhichmostmetabolismoccurs of this alkaloid. Although recent studies have con-
although small but insignificant contributions from tributed greatly to understanding the molecular ge-
lung and kidney also having been identified. One netics underlying the diversity of different isoforms
routeofmetabolisminvolvesesterasecleavagewhich of the flavin monooxygenases in various organs and
releasesthenecineandnecicacidmoietiesneitherof tissuesofanumberofspeciesthereisasyetnoclear
which are toxic or undergo further metabolism. This indication as to the molecular basis of substrate
A.S.Prakashetal.rMutationResearch443(1999)53–67 57
selectivity of the different forms for any of the with2Band2Disoformsalsohavingthisactivity.In
pyrrolizidine alkaloids. human liver microsomes early immunochemical in-
A number of reports show that microsomal cy- hibition studies, using antibodies raised in rabbits
tochrome P450 monooxygenases may also be re- against purified rat liver CYP isoforms, suggested
X
sponsible for catalysing the N-oxidation of some the debrisoquine 4-hydroxylase contributes signifi-
pyrrolizidine alkaloids in some species. In rats for cantlytooxidationoflasiocarpineandmonocrotaline
example senecionine N-oxidase activity is markedly w37x and more recently strong evidence has accumu-
genderdifferentiated,thisconversionoccurringmuch lated showing the role of CYP3A4 in toxification of
morerapidlyinmalethaninfemaleanimals,andthis senecionine by the dehydrogenation pathway w36x.
was attributed to the higher activities of the ‘male Importantly, CYP3A4 was also shown to be able to
specific’ liver cytochrome P450 isoform UT-A. An- catalyse senecionine N-oxide formation w36x, which
other CYP isoform, PCN-E, induced by treatment of strongly implicates this single enzyme as simultane-
the animals with the anti-glucocorticoid preg- ously catalysing toxification and detoxification of
nenolone 16a-carbonitrile was also active in this this alkaloid in exposed individuals. Interestingly,
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regard 32. the abundance of this enzyme in liver varies over a
These rat liver enzymes are now identified in the 30-fold range between individuals which confound
nomenclature system based on primary amino acid attempts to make predictions regarding the rate and
sequence homology as CYP2C11 and CYP3A1 extent of metabolism of this alkaloid by either path-
(cid:14)http:rrdrnelson.utmem.edurnelsonhomepage.html.. way in any individual. A similar dual role (cid:14)toxifica-
A CYP isoform in the 2C subfamily isolated from tion and detoxication. for the mixture of CYP3A
guinea pig liver also showed N-oxidase activity to- subfamily enzymes in male rat liver induced by
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wards senecionine 35. The human liver enzyme dexamethasone treatment had been reported
CYP3A4 has also been identified as having senecio- previously with senecionine metabolism w33x and
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nine N-oxidase activity 36. The abundance of this equivalent findings with female rats treated with
enzyme varies widely between individuals which spironolactone (cid:14)which also induced CYP3A subfam-
suggeststhat interindividualvariationin clearance of ily isoforms. have also been reported more recently,
this alkaloid may be very variable but this would again with senecionine as the alkaloid under study
also depend on the substrate selectivities of the w38x. In a recent paper, rat CYP3A enzymes were
human flavin monooxygenases which appear to be also identified as catalysing 14C labeled monocro-
undetermined. taline bioactivation which resulted in covalent bind-
Conversion of PAs to reactive toxic pyrrolic ing of 14C to liver microsomes w39x.
metabolites is now well established to be due to
a-carbon oxidation (cid:14)dehydrogenation. catalysed by
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cytochromeP450monooxygenases 28.Theprimary 5. Toxicity in animals
oxidised metabolites are reactive and undergo spon-
taneous conversion to electrophilic species which 5.1. Li˝estock poisonings
can undergo Michael addition reactions with cellular
nucleophiles. The sacrificial (cid:14)protective. nucleophile Although grazing animals do not naturally forage
reducedglutathione(cid:14)G-SH.trapssomeofthesereac- on PA containing plants, they are consumed in
tive products as a detoxication route to their clear- drought periods when other food is in short-supply
ance but critical protein and nucleic acid nucle- or if the feed-stock is contaminated. Substantial dif-
ophiles also react yielding adducts which have been ferences in susceptibility occur between animals of
proposed to cause cell toxicity (cid:14)see Section 8.. different species. Pigs and poultry are most suscepti-
Rat, human, and guinea pig tissues and enzyme ble, while horses and cattle are less so but sheep and
preparationshavebeenmostcommonlyusedtochar- goats are relatively resistant to PA toxicity. These
acterise the enzymes catalysing these bioactivation differences are believed to be due partly to the
(cid:14)toxification. reactions with the most commonly variationsintheefficiencywithwhichliverenzymes
identified isoforms being in the CYP3A subfamily metabolise the parent alkaloid to the toxic pyrrole
58 A.S.Prakashetal.rMutationResearch443(1999)53–67
and with respect to sheep, partly by enzymes in the Some of the plant species known to cause cancer in
rumen w40x. rodents are S. longilobus, w55x Petasites japanicus
Several workers have reported PA poisoning of Maxim w56x, Tussilago farfara L. w57x, Symphytum
pigs, poultry and ducks in Australia w41–43x. In the officinale w58x, Farfugium japonicum w59x, Ligularia
mostrecentcase,Gauletal.w44xrecordedacontami- dentata w60x and S. cannabifolis w61x. Further, indi-
nation of feed stocks with poisoning in pigs, poultry vidualPAcompoundssuchasmonocrotalinew62,63x,
and calves in southern Australia when higher than heliotrine w64x, lasiocarpine w65,66x, clivorine w60x,
average summer rainfall aided the growth of he- petasitenine w67x and riddelliine w68x have also been
liotrope weed in wheat fields. shown to be carcinogenic in experimental animals.
Serious outbreaks of PA poisoning in cattle have
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occurred throughout the world 45–47. These epi-
demics generally occur after a period of high winter 6. Human poisonings
rainfall followed by a dry summer, conditions which
favour the growth of PA containing weeds in the 6.1. Hepatic ˝eno-occlusi˝e disease (VOD)
grazing pasture. There have also been sporadic cases
of poisoning due to the contamination of hay with VOD characterised by epigastric pain with ab-
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leaves and seeds of toxic plants 48,49. Calves and dominaldistensionduetoasciteshasbeenassociated
young animals show higher susceptibility than older with human consumption of PAs often by the acci-
cattle, most of the animals involved in the epidemic dental contamination of grain with seeds containing
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in 1994 being less than 3 years old 46. PA poison- PA or through herbal remedies. Some of the most
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ing has been reported in yaks in Bhutan 50. In serious outbreaks of PA poisoning were reported in
addition to the other characteristic features of PA NW Afghanistan w69x and central India w4x following
toxicity these animals suffered from skin lesions a severe period of drought, during which he-
with hyaline parakeratosis. Sulphur bound pyrrolic liotropium plants were seen to thrive in the region.
adducts were demonstrated in formalin fixed liver The staple food was contaminated with seeds of the
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51 and bound to haemoglobin in the circulating H. popo˝ii plant. The most recent outbreak occurred
blood w52x of affected animals. in 1992 in Tadjikistan w5x. These seeds contained the
PAs, heliotrine and lasiocarpine. VOD was endemic
5.2. Toxicity in laboratory animals in regions of South America during the latter part of
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the century 70 but with better education on the
5.2.1. Lung toxicity proper identification of plants this is no longer the
Pulmonary lesions produced by PAs have been case. However, sporadic cases are still being re-
extensively investigated, mainly in rats, but also in ported from around the world w71–73x.
non-human primates. In one study, dogs dosed with
60 mgrkg of monocrotaline by body weight pro- 6.2. Teratogenicity
duced ultrastructural changes in endothelial cells of
the alveolar capillaries, prominent accumulation of VOD has been reported in an infant born to a
platelets and the appearance of interstitial oedema woman who had consumed herbal tea brewed from
w53x. Similar lesions were observed in Sprague– the leaves of T. farfara (cid:14)coltsfoot. which contained
Dawley rat lungs using a single subcutaneous injec- 0.6 mgrkg senecionine by dry weight w74,75x.
tion of monocrotaline at 60 mgrkg by body weight At least one of the components in S. madagas-
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54. cariensis, an introduced species, which has spread
over vast regions of coastal South East Queensland
5.2.2. Carcinogenicity inAustraliaseemstohavehighlipophilicityanditis
While there is no evidence of cancer in the litera- suggested that this may enable it to cross the pla-
ture concerning domestic animals exposed to PAs, centa and cause hepatic failure in the foetus. This
studies carried out under laboratory conditions have view is supported by the recent observation of
been able to produce PA-induced cancer in rodents. pyrrolizidine alkaloidosis in a two-month-old foal
A.S.Prakashetal.rMutationResearch443(1999)53–67 59
caused by consumption of S. madagascariensis by presence of hepatic megalocytes. Field experiments
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the mother 76. have demonstrated megalocytosis in the livers of
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livestockfedPAcontainingplantmaterial 10,83,84.
6.3. Carcinogenicity In addition megalocytes have been observed in ex-
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perimental animals, rats 7,85,86, mouse, sheep,
Schoental’sgroup which showed the formation of horse, pig and most recently in the chick embryo
primary liver tumours in rats following feeding of w87x. Mattocks w26x suggested that these cells appear
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alkaloids 77 first raised the possibility that PAs within a few weeks and this lesion may persist for
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may also play a role in human carcinogenesis 78. the lifetime of the animal. However, recently it was
Since then several PAs and their metabolites have shown that transplantation of normal hepatocytes
been shown to be carcinogenic in rodents. However, into rats treated with lasiocarpine significantly re-
though there are several recorded cases of human duced the number of existing megalocytes w86x.
exposure to PAs, with exposure levels ranging from Megalocytosis has been demonstrated in mam-
acute to chronic levels, there exist no reports to date malian cell culture also. It was seen in cultured
of cancer associated with such exposures. bovine pulmonary artery endothelial cells (cid:14)BEC. ex-
posed to a monocrotaline pyrrole (cid:14)MCTP. w88x. Kim
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et al. 89 werealsoableto demonstratemegalocyto-
7. Toxicology of pyrrolizidine alkaloids sis in a bovine kidney epithelial cell line exposed to
a range of alkaloids. Megalocytosis can be produced
The classic feature of chronic PA poisoning is by a single sublethal dose w90x or by a cumulative
VOD, hepato-splenomegaly and emaciation. After effect of small doses w91x.
the liver, the lungs are the next most common sites Megalocytosis has been found in other organs
ofPAtoxicity.Pyrrolesformedbythemetabolismin such as kidney and lungs as well w92x. It has been
the liver can travel to the lungs. Initial changes seen shown to occur in cultured human fetal liver cells
in the pulmonary vasculature included thrombi in w93x but has never been observed in the affected
vessels, acute inflammation and thickening of vessel human livers w3x.
walls leading to occlusion. These effects along with It is believed that the formation of megalocytosis
the interalveolar septal fibrosis lead to pulmonary is a result of the action of the metabolite pyrrole
hypertension. The result of the impaired pulmonary ester w26x. It is thought that the pyrrole disrupts the
blood flow is increased work for the right ventricle cell cycle by damaging key genes which control cell
causing it to hypertrophy and eventually leading to division leading to mitotic bypass w94x. Section 9
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congestive heart failure 79. addressesthequestionofPAeffectsonthecellcycle
Other important chronic toxicities of PAs are in greater detail.
antimitotic effects observed in rodents and domestic
animals and cancer reported only in rodents. 7.2. Genetic toxicology of pyrrolizidine alkaloids
7.1. Antimitotic acti˝ity Clark was able to classify several PAs according
to their mutagenicity in Drosophila melanogaster
One of the characteristic features of chronic w95x. Monocrotaline, lasiocarpine and heliotrine (cid:14)see
pyrrolizidine alkaloid poisoning in animals is the Fig. 1. showed strong mutagenic property in this
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developmentofenlargedcells,ormegalocytosis 80. assay.Numerousotherstudieshavedemonstratedthe
Megalocytes appear to be the result of a combined mutagenicity of PAs w96–99x. Milk from lactating
action of PAs on the hepatocytes, a stimulus to rats fed PAs was shown to be mutagenic in the
regenerate following parenchymal cell injury, and Salmonellarmammalian-microsome mutagenicity
the antimitotic action of the pyrrole metabolites of test w10x. Recently, Berry et al. w100x used a primary
w x w x
PAs 26. Post-mortem examinations of cattle 81 hepatocyte-mediated V79 cell mutagenesis and
w x w x
horses 81,82 and yaks 51 which have died after DNA-repair assay system to study the genotoxic
consuming PA containing plants have revealed the effectsof PAs. Basedon theirresultstheyconcluded
60 A.S.Prakashetal.rMutationResearch443(1999)53–67
thatriddelliineandstructurallyrelatedPAsarelikely DNA–protein crosslinking was significantly greater
to be hepatocellular carcinogens as well as cytotoxic than DNA–DNA crosslinking. Despite replacement
agents. with fresh medium lacking MCTP every two days,
the crosslinking factor remained elevated till day 10.
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Hinksetal. 107 alsoshowedDNA–proteininterac-
8. Interactions of pyrrolizidine alkaloids with tions predominate over DNA–DNA ones in cultured
cellular components MadinDarbybovinekidney(cid:14)MDBK. epithelialcells
exposed to a range of eight bifunctional alkaloids in
The highly electrophilic nature of PA metabolites the presence of an external metabolising system.
suggests that they would react readily with nucleo- This study also showed a correlation between cross-
philic tissue constituents such as DNA and proteins. linking ability and suppression of colony formation
The earliestobservationof PA interactingwithDNA that strengthens the hypothesis that crosslinking is
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was reported by White et al. 101. Pyrroles alkylate involved in the biological activity of PAs. Some of
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proteins as well 102. Chromatography, NMR and the most potent crosslinkers were able to inhibit
spectral analysis were used to show interaction be- colonyformationaltogether.Laterthisgroupdemon-
tweentheC-7positionofdehydroretronecine(cid:14)DRN., strated megalocytosis in cultured cells exposed to
a metabolite of monocrotaline, and the sulfhydryl these alkaloids w89x (cid:14)see below.. The most recent
groups of cysteine and glutathione. Alkylation be- work in MDBK cells w108x suggests a DNA–PA–
tween the C-7 of DRN and the exocyclic amino protein structure. The protein isolated had a molecu-
group (cid:14)NH . of deoxyguanosine (cid:14)dG. has also been lar weight of 40–60 kDa and a net acidic charge
2
demonstrated w103x. Even though DRN is a bifunc- (cid:14)isoelectric point 4.2–5.0.. The authors suggest it
tional alkylating agent only monoadducts with may be actin (cid:14)pI 5.4, molecular weight 45 kDa..
equimolar quantities of DRN and dG were detected
and the C9 was less reactive than the C7 position in
this study. In vitro studies carried out in our lab 9. Effect of pyrrolizidine alkaloids on the cell
showed that dehydromonocrotaline(cid:14)DHM. alkylated cycle
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N7 guanine in a sequence selective fashion 104.
Further, we also found evidence that DHM formed In yeast, damage to DNA results in cell cycle
polymers at sub millimolar concentrations which in- arrest at one of the checkpoints, enabling DNA
duced multiple DNA fragment crosslinks, a phe- repair. This happens in mammals too, however, an
nomenon never observed before with any other class additional cell death (cid:14)apoptosis. pathway may be
of DNA crosslinking agents. Polymer formation by activated, since the primary aim of multicellular
bifunctionalPA metabolites in cells has not yet been organisms is the survival of the whole, rather than
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reported, but Mattocks 26 predicted its formation the preservation of individual cells which may foster
and discussedthe relevance of these structures under mutations.
biological conditions. Theroleofthep53tumoursuppressorgeneinthe
Petry et al. w105x used the alkaline elution tech- control of the cell cycle at the G1rS stage is well
nique to show dose dependent DNA–DNA inter- established. It has been suggested that p53 may be
strand and DNA–protein crosslinks in hepatic nuclei involved in controlling the cycle at G2rM phase as
ofSprague–Dawleyratstreatedintraperitoneallywith well w109x. Other important cell cycle regulatory
5–30 mg monocrotalinerkg body weight. The alka- elements involved in G2rM and MrG1 checkpoints
line elution was also used to determine crosslinking are rumq1, cdc2, cdc25, cyclin B, and RMSA-1
inculturedporcinepulmonaryarteryendothelialcells (cid:14)regulator of mitotic spindle assembly, w110x. Re-
(cid:14)PEC. exposed to an unspecified monocrotaline pyr- cently, Couet et al. w111x observed a specific muta-
role (cid:14)MCTP. w106x. DNA–DNA and DNA–protein tion in codon 249, exon 7 of the p53 gene in a
crosslinking were seen 4 h after exposure to MCTP human Chang liver cell line treated with PAs and an
and the degree of crosslinking increased till the externalmetabolisingsystem.Recentworkinourlab
medium was changed at 48 h. By this time the w112x using SD rats treated intraperitoneally with
A.S.Prakashetal.rMutationResearch443(1999)53–67 61
monocrotaline at 65 mgrkg for 4 weeks showed the without intervening mitosis. Contrary to this view,
formation of moderate to extensive regions of mega- however, PA-induced G2rM arrest in cell culture
locytotic parenchymal cells in the livers in five out has been reported recently w116,117x. However, it is
offivefemalesbutnotinthemales(cid:14)0r2.. Threeout not clear whether the authors considered mitosis
of these five females showed mutations in the codon bypass as a possibility.
152 in exon 5 of the p53 gene. Based on our understanding, we suggest that PA-
It has been proposed that megalocytosis might be induced megalocytosis may be due to DNA damage
due to mitosis bypass leading to continual synthesis leading to mutation in cell cycle regulatory genes
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of DNA and proteins 113. Thus, while DNA dam- and subsequent altered cell cycle regulation such as
aging agents (cid:14)e.g., doxorubicin, X-radiation. in gen- over-expression of the rum protein.
eral lead to cell cycle arrest at G2rM phase PAs
have the ability to allow the cells to bypass mitosis.
Inthisregard,itispertinenttopointoutthatinyeast 10. Mechanism of pyrrolizidine alkaloids toxicity
(cid:14)S. pombe., an over-expression of the rum protein
(cid:14)replication uncoupled from mitosis. p25rumq1 The various routes by which PA can affect hepa-
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leadstosuchamitoticbypassinthissystem 114.A tocytes are depicted diagrammatically in Fig. 4.
similar situation was reported in human cells by PA esters pass into the hepatocytes via the sinu-
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Waldmanetal. 115 whodemonstratedinvitrowith soidal blood. In the hepatocyte the PAs are
p21 deficient human colorectal cancer cells, which metabolised via three major routes. The ester alka-
whenexposedtoavarietyofDNAdamagingagents, loid may undergo hydrolysis through esterase activ-
arrested in G2 then underwent additional S phases ity or else oxidation via the microsomal mono-
Fig.4.RepresentativeschemeofPAmechanismsoftoxicity:PA,pyrrolizidinealkaloid;EPy,pyrroleester;APy,pyrrolicalcohol;GSH,
glutathione;Py-SG,pyrrole-glutathioneconjugate;Py-SPr,pyrroleboundtoproteinthiol;RBC,redbloodcell.ThedashedlinesforAPy
indicatethatitisaminormetaboliteresponsibleforchroniceffectsbecauseofitslowerreactivityandlonghalf-life.
62 A.S.Prakashetal.rMutationResearch443(1999)53–67
oxygenases to either N-oxides or dehydro PAs or heart where they may cause damage to the macro-
pyrroles w26x. Hydrolysis and N-oxide formation are molecules of these organs w120x. The immediate
detoxication reactions and as such are generally reactive effects of the primary toxic metabolites are
without harm to the cell. Dehydro PAs are consid- considered to be responsible for the damage to the
ered to be the primary toxic metabolites w26x and periacinar hepatocytes (cid:14)because the activating P450
mayreactwithavailablenucleophileswithinthecell. enzymes are concentrated in the cells of this
Theseesterpyrroles(cid:14)EPy. may alsoundergohydrol- parenchymal zone. and to the associated sinusoidal
ysis with the formation of pyrrolic alcohols (cid:14)APy.. liningand wallsof the smallhepaticveinsleadingto
These are the secondary toxic metabolites and while VOD.
they are far less reactivethan the ester pyrroles, they With outbreaks of acute PA toxicities in humans
are far more persistent. it is estimated that about 20% die and some 50%
The alkylating species is thought to be a carbo- recover completely within a few weeks. Of the re-
nium ion with its reactive centre at C9. If the C7 mainder some 20% appear to recover clinically but
position also has an oxygen function then active maydevelopchronicVODandcirrhosisafterseveral
centres exist at both C7 and C9 with the C7 position years.Othersdevelopasub-acuteVODandthismay
the more active. This enables this type of toxic eventually resolve or else progress to chronic VOD
metabolite to act as a bifunctional alkylating agent and cirrhosis w121x. There are also reports of PA
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26. toxicities in domestic animals in which following
Thepyrroleonceformedwithinthe cell maybind relatively low doses of the alkaloids, often insuffi-
covalently to sulphur, nitrogen and oxygen contain- cient to cause acute toxicity, death due to chronic
ing groups on various macromolecules. While the hepatic failure occurs several months or years later
S-boundpyrrolesare themoststableandmaypersist w122x.
in the cell for a considerable time as protein-bound It has been established that following a single
complexes (cid:14)Py-SPr., these reactions are by and large dose of a PA, almost all of the compound including
reversible. This means that the pyrrole moiety may its soluble metabolites is eliminated from the body
be released from its bond to protein into the cell as within 24 h w123x. Yet in some individuals such an
APy with secondary toxicity. exposure can lead to a progressive and eventually
The production of the primary toxic metabolite is total hepatopathy w90x. It has been suggested that the
followed by reaction mainly with proteins at the site presence of reversibly bound pyrrolic metabolites in
offormation.Thesepyrrolestovaryingdegreeshave the hepatocytes and endothelial cells of the liver are
persistence in aqueous media which enables them to responsible for this effect w124x. If re-released
penetrate into the nucleus and react with DNA caus- pyrrolesreact with GSH they will be safely removed
ing crosslinking within DNA and between DNA and from the liver over time. If re-released pyrroles go
nucleo-proteins. These reactions lead to immediate on to bind to vital macromolecules then hepato-
damage to the hepatocyte. Reactions with soluble cellular necrosis and VOD may be sustained. If the
molecules in the cytoplasm such as GSH are, how- pyrroles bind to DNA then mutations either leading
ever, protective for the cell. GSH-pyrrole adducts to an antimitotic action andror cancer may occur.
(cid:14)Py-SG. and other soluble reaction products are The agents re-released from transiently ineffective
eliminated from the cell into the bile andror sinu- macromolecularbinding would be the persistent sec-
soidal blood for excretion into the urine. ondarytoxicmetabolitepyrrolicalcohols.Thesehave
Primary toxic metabolites of many PAs are suffi- the persistencein the body and the potential to reach
ciently persistent as to be able to pass from the all parts of the liver cell and its surrounding struc-
hepatocyteinto the adjacentDisse´ space and into the turesandareconsideredtobemainlyresponsiblefor
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sinusoidallumen 118. Here the pyrrolesmay attack targeting specific DNA binding sites, leading to the
the associated sinusoidal lining cells such as en- promotionofthemegalocytosisintheliverandother
dothelialcellsandalsobecomeboundonpassingred tissues, a radiomimetic-type effect w26x.
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blood cells 118,119. Specially long lived primary Chronic hepatotoxicity caused by PA exposure
pyrrolic metabolites may even reach the lungs and will be associated with hepatocellular injury leading