genotoxicity of pyrrolizidine alkaloids

J. Appl. Toxicol. 2010; 30: 183–196 Published in 2010 by John Wiley & Sons, Ltd.

Review

Received 7 October 2009, Accepted 16 November 2009, Published online in Wiley InterScience: 28 January 2010

(www.interscience.wiley.com) DOI 10.1002/jat.1504

Genotoxicity of pyrrolizidine alkaloids†

Tao Chen,* Nan Mei and Peter P. Fu

ABSTRACT: Pyrrolizidine alkaloids (PAs) are common constituents of many plant species around the world. PA-containing plants are probably the most common poisonous plants aff ecting livestock and wildlife. They can infl ict harm to humans through contaminated food sources, herbal medicines and dietary supplements. Half of the identifi ed PAs are genotoxic and many of them are tumorigenic. The mutagenicity of PAs has been extensively studied in diff erent biological systems. Upon metabolic activation, PAs produce DNA adducts, DNA cross-linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations and chromosome mutations in vivo and in vitro. PAs induced mutations in the cII gene of rat liver and in the p53 and K-ras genes of mouse liver tumors. It has been suggested that all PAs produce a set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine-derived DNA adducts and similar types of gene mutations. The signature types of mutations are G : C → T : A transversion and tandem base substitutions. Overall, PAs are mutagenic in vivo and in vitro and their mutagenicity appears to be responsible for the carcinogenesis of PAs. Published in 2010 by John Wiley & Sons, Ltd.

Keywords: pyrrolizidine alkaloid; genotoxicity; mutation; DNA damage; carcinogenesis; mutational signature

*Correspondence to: Tao Chen, HFT-130, 3900 NCTR Rd, Jeff erson, AR 72079, USA.

Email: [email protected]

This article is a US Government work and is in the public damain in the USA.

National Center for Toxicological Research, US Food and Drug Administration,

Jeff erson, AR 72079, USA

†The views presented in this article do not necessarily refl ect those of the US Food

and Drug Administration.

INTRODUCTION

Occurrence and Toxicity

Pyrrolizidine alkaloids (PAs) are constitutively formed in many

plant species around the world. More than 660 PAs and PA

N-oxides have been identifi ed in over 6000 plants mainly con-

tained in the Boraginaceae, Compositae and Leguminosae fami-

lies (Roeder, 1995, 2000; Stegelmeier et al., 1999). About half of

these PAs formed are toxic. Therefore, The PA-containing plants

are poisonous to livestock and wildlife and have caused tremen-

dous livestock loss (Arzt and Mount, 1999; de Lanux-Van Gorder,

2000; Fletcher et al., 2009; Fowler, 1968; Knight et al., 1984;

Seaman, 1978, 1987; Sharrock, 1969; van der Watt et al., 1972;

Wiltjer and Walker, 1974). PAs are also the leading plant toxins

associated with disease in humans through contamination of

staple foods, honey, milk, herbal teas and herbal medicines

(Arseculeratne et al., 1981, 1985; Bach et al., 1989; Bah et al., 1994;

Culvenor et al., 1981; Deinzer et al., 1977; Dickinson et al., 1976;

Edgar et al., 1992, 2002; Huxtable et al., 1986; Jago, 1969; Kumana

et al., 1983: 1985; Mattocks, 1980; Ridker et al., 1985; Roitman,

1981; Steenkamp et al., 2000; White et al., 1984; Zhao et al., 1989).

Chemical Structure

PAs are ester alkaloids composed of a necine (two fused fi ve-

membered rings joined by a single nitrogen atom) and a necic

acid (one or two carboxylic ester arms) (Fig. 1). Toxic PAs are

esters of unsaturated necines having a 1,2 double bond.

Structures of several representative carcinogenic PAs are shown

in Fig. 2. Riddelliine, retrosine, monocrotaline and symphytine

are retronecine-type PAs; lasiocarpine and heliotrine are heliotri-

dine-type PAs; and senkirkine and petasitenine are otonecine-

type PAs. In contrast to otonecine-type PAs that contain

monocyclic necines, retronecine- and heliotridine-type PAs have

bicyclic necine bases and they are enantiomers each other at

the C7 position, the retronecine-type PAs possessing an R abso-

lute confi guration and the heliotridine-type PAs with an S

stereochemistry.

Metabolism

As with many other xenobiotics, PAs require biotransformation

to introduce reactive or polar groups so that they can be conju-

gated to form polar metabolites for excretion out of the body.

There are three principal pathways for PA metabolism, including

hydrolysis of PAs to release necines and necic acids, N-oxidation

to form PA N-oxides and oxidation of PAs to produce dehydropyr-

rolizidine (pyrrolic ester) derivatives (Fig. 3). The liver is the main

organ for PA metabolism, although metabolism in other tissues

has also been identifi ed (Lafranconi and Huxtable, 1984).

When PAs are absorbed into tissues like liver and lung through

blood, some of them are cleaved into necines and necic acids by

nonspecifi c esterases. The necines and necic acids are not toxic

and the necines may undergo further conjugation to be excreted

via the kidneys and urine (Roeder, 1995). This metabolic pathway

is an important detoxifi cation route. Rats are very susceptible to

toxicity of PAs, partially if not totally, due to lack esterase activity

in their livers, whereas guinea pigs possess marked resistance to

the toxic eff ects of PAs because of their particularly high liver

esterase activity (Dueker et al., 1992).

Retronecine- and heliotridine-type PAs can become PA

N-oxides via N-oxidation of the necine bases while otonecine-

type PAs cannot form PA N-oxides because the nitrogen in their

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www.interscience.wiley.com/journal/jat Published in 2010 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 2010; 30: 183–196

Retronecine-type

Heliotridine-type Otonecine-type

N

O CH2

O

OCH2OHHOH

H3C

OCH2

H

RiddelliineN

O CH2

OH

O

CH2OHHOH

H3C

OH

RetrorsineN

O CH2

O

CH3

O

CH3OH

O

HOCH3

H

H

Monocrotaline

N

O CH2

O

CH3OH

H

OHC(CH3)2HO

H3C

OH

H3C

Symphytine

N

O

O

CH3OCH3

H

O

C(CH3)2OHHO

H3C

OH

H3C

LasiocarpineN

CH2

O

O

HC(CH3)2HO

H3C

HHO

OCH3

H

Heliotrine

N

O CH2

OH

O

CH3HO

H3C

O

CH3

O

senkirkine

N

O CH2

OH CH3

O

CH3HO

H2C

O

CH3

O

O

Petasitenine

CH2

CH3

CH3

Figure 1. Schematic structure of pyrrolizidine alkaloid.

Figure 2. Structures of representative carcinogenic pyrrolizidine alkaloids.

necine base is methylated. PA N-oxides are generally regarded as

detoxifi cation products because the metabolites can be con-

jugated for excretion (Williams et al., 1989a, b). However, PA

N-oxides can be metabolically converted back to their parent PAs

to produce toxic/tumorigenic eff ects if their parent PAs are toxic/

tumorigenic (Chou et al., 2003b; Mattocks, 1971; Wang et al.,

2005c; Yan et al., 2008).

Pyrrolic esters are produced through hydroxylation of the

necine base of retronecine- and heliotridine-type PAs at the C3

and C8 position to form 3- or 8-hydroxynecine derivatives fol-

lowed by spontaneous dehydration. For otonecine-type PA,

pyrrolic esters are generated through oxidative N-demethylation

of the necine base followed by ring closure and dehydration (Fu

et al., 2004). Pyrrolic ester metabolites are very reactive and can

bind to one or two molecules of glutathione to form glutathione

conjugates for excretion. They also can bind to DNA and proteins

to generate DNA adducts, protein adducts and DNA and protein

cross-links. Thus, metabolic formation of pyrrolic ester metabo-

lites has been considered as the primary metabolic activation

for the genotoxicity and carcinogenicity of PAs. In addition, less

reactive longer-lived (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-

5H-pyrrolizine (DHP) produced by hydrolysis of pyrrolic esters

is a considered secondary toxic metabolite due to its antimi-

totic, mutagenic and carcinogenic eff ects (Fu et al., 2004; Prakash

et al., 1999).

Carcinogenicity and Hepatotoxicity

Many PA-containing plants and individual PA compounds have

been tested in animal models and shown to be carcinogenic in

diff erent tissues. The liver is the main carcinogenic target.

However, tumors induced by PAs also were found in lung, kidney,

skin, bladder, brain and spinal cord, pancreatic islets and adrenal

gland (Allen et al., 1975; Brandange et al., 1970; Chan et al., 1994;

Cook et al., 1950; Furuya et al., 1976; Harris and Chen, 1970;

Hirono et al., 1976, 1977, 1978, 1979, 1983; Johnson et al., 1978;

Kuhara et al., 1980; Mattocks and Cabral, 1982; Mori et al., 1984;

Peterson et al., 1983; Rao et al., 1983; Rao and Reddy, 1978;

Schoental, 1975; Schoental and Cavanagh, 1972; Schoental et al.,

1954, 1970, 1971; Schoental and Head, 1957; Shumaker et al.,

1976; Svoboda and Reddy, 1972, 1974; Williams, 1970). Despite

no clear evidence that PAs induce tumors in humans, the fre-

quent occurrence of primary liver tumors in the natives of Central

Africa and South Africa has been associated with the consump-

tion of traditional medicinal PA-containing plants (Pavlica and

Samuel, 1970; Schoental, 1968; Schoental and Coady, 1968;

Williams et al., 1967). It has been reported that PA-containing

plants induce hepatic veno-occlusive disease in humans. This

disease is regarded as specifi c for PA intoxication. The clinical

symptoms usually occur suddenly and include vomiting, enlarge-

ment of the liver and bleeding diarrhea. Children are more sensi-

tive to PA intoxications than adults (Bach et al., 1989; Bras et al.,

1954; Kumana et al., 1985; Mohabbat et al., 1976; Ortiz Cansado

et al., 1995; Ridker and McDermott, 1989; Ridker et al., 1985;

Schoental and Coady, 1968; Sperl et al., 1995; Tandon et al., 1976;

Weston et al., 1987; Yeong et al., 1990).

In this article, we review and update information on the geno-

toxicity and mutagenicity of PAs, including primary DNA damage,

chromosome damage and mutations induced by PAs or PA-

containing plants. For detailed information concerning the

chemical and physical properties, metabolism, hepatotoxicity,

carcinogenicity and other toxicities of PAs, readers are referred to

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Genotoxicity of pyrrolizidine alkaloids

J. Appl. Toxicol. 2010; 30: 183–196 Published in 2010 by John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jat

books and other review articles on PAs and PA-containing plants

(Cheeke, 1988; Fu et al., 2004; IARC, 1976; IPCS, 1988; Mattocks,

1986; Prakash et al., 1999; Robins, 1984; Roeder, 1995, 2000;

Stegelmeier et al., 1999).

Primary DNA Damage Induced by PAs

DNA adducts

Metabolism of PAs in vitro and in vivo generates DHP-derived

DNA adducts (Table 1). Candrian et al. (1985) treated rats of both

sexes with tritiated senecipylline and senecionine and deter-

mined covalent binding of the alkaloids to DNA using HPLC/

radioactivity analysis of hydrolyzed DNA 6 h or 4–5 days after the

treatment. They found PA-derived DNA adducts in rat livers,

lungs and kidneys. The DNA damage was induced 6 h after treat-

ment and persisted during the following 4 days. However, the

DNA adducts were not isolated and characterized.

To identify the types of DNA adducts induced by PAs, Fu and

his research group developed a 32P-postlabeling/HPLC method

and determined the DNA adduct formation in vitro and in vivo

using riddelliine as a model PA (Chou et al., 2003a; Xia et al., 2003;

Yang et al., 2001). A set of DHP-derived DNA adducts were iden-

tifi ed, among which two were enantiomers of DHP-derived

7′-deoxyguanosin-N2-yl adducts and the other six were DHP-

modifi ed dinucleotides. Further studies showed that diff erent

types of tumorigenic PAs and PA-containing plants induced the

same set of DHP-derived DNA adducts in vivo and in vitro. Those

PAs and PA-containing plants included clivorine, heliotrine, lasio-

carpine, monocrotaline, retrosine, comfrey root extract, comfrey

compound oil, coltsfoot root extract, Flos farfara extract and

Ligularia hodgsonnii extracts (Chou and Fu, 2006; Wang et al.,

2005a, b; Xia et al., 2004, 2006, 2008). Therefore, it is suggested

that the formation of DHP-derived DNA adducts is common to

all types of tumorigenic PAs.

The levels of the DHP-derived DNA adducts correlated closely

with tumorigenic potency in the rats fed diff erent doses of rid-

delliine (Chou et al., 2003c; Yang et al., 2001). Riddelliine mainly

induced liver hemangiosarcomas in rats and mice (Chan et al.,

2003). Liver contains two types of cells involved in tumorigenesis,

endothelial and parenchymal cells, and hemangiosarcomas

develop from endothelial cells. F344 rats and B6C3F1 mice were

treated by gavage 5 days a week for 2 weeks with riddelliine at

1.0 mg/kg for rats and 3.0 mg/kg for mice. The treatment resulted

Figure 3. Metabolism of pyrrolizidine alkaloids.

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Table 1. Primary DNA damage induced by pyrrolizidine alkaloids

Agent Testing system Result Reference

DNA adductsComfrey root extract/

comfrey compound oilIn vivo 32P-postlabeling/HPLC

analysis DHP-derived DNA adducts were

formed in rat liverChou and Fu (2006)

Clivorine In vitro 32P-postlabeling/HPLC analysis with metabolic activation

DHP-derived DNA adducts were formed in vitro

Xia et al. (2004)

Coltsfoot root extract In vivo 32P-postlabeling/HPLC analysis

DHP-derived DNA adducts were formed in rat liver

Chou and Fu (2006)

Flos farfara Extract In vivo 32P-postlabeling/HPLC analysis

DHP-derived DNA adducts were formed in rat liver

Chou and Fu (2006)

Heliotrine In vitro 32P-postlabeling/HPLC analysis with metabolic activation

DHP-derived DNA adducts were formed

Xia et al. (2008)

Ligularia hodgsonnii extracts

In vitro 32P-postlabeling/HPLC analysis with metabolic activation


Xia et al. (2004)

Lasiocarpine In vitro 32P-postlabeling/HPLC analysis with metabolic activation


Xia et al. (2006)

Monocrotaline In vivo or in vitro with metabolic activation 32P-postlabeling/HPLC analysis

DHP-derived DNA adducts were formed in vitro and in rat liver

Wang et al. (2005b)

Retrorsine In vivo or in vitro with metabolic activation 32P-postlabeling/HPLC analysis

DHP-derived DNA adducts were formed in vitro and in rat liver

Wang et al. (2005a)

Riddelliine In vivo or in vitro with metabolic activation 32P-postlabeling/HPLC analysis

Two enantiomers of DHP-7-deoxyguanosin-2N-yl adducts and DHP-modifi ed dinucleotides were formed in vitro and in rat liver

Chou et al. (2003a–c, 2004); Xia et al. (2003); Yang et al. (2001)

Senecionine In vivo covalent binding analysis in rat using 3H labeling

Uncharacterized DNA adducts were identifi ed in rat liver, lung and kidney

Candrian et al. (1985)

Seneciphylline In vivo covalent binding analysis in rat using 3H labeling

Uncharacterized DNA adducts were identifi ed in rat liver, lung and kidney

Candrian et al. (1985)

DNA cross-linkingHeliosupine In vitro cells DNA–DNA crosslinks Hincks et al. (1991)Jacobine Alkaline elution in rat liver DNA–DNA crosslinks

DNA–protein crosslinksPetry et al. (1986)

Latifoline In vitro cells DNA–DNA crosslinks Hincks et al. (1991)Monocrotaline

(Dehydromonocrotaline)In vitro cross-linking assay of

PA-exposed cells or pyrrolic PA-exposed nuclei

In vivo and in vitro alkaline elution assay

DNA–DNA crosslinksDNA–protein crosslinks

Coulombe et al. (1999); Kim et al. (1995); Pereira et al. (1998); Petry et al. (1984); Petry and Sipes (1987); Rieben and Coulombe (2004); Tepe and Williams (1999); Wagner et al. (1993); Weidner et al. (1990)

Retrorsine (dehydroretronecine)

In vitro cross-linking assay of PA-exposed cells or pyrrolic PA-exposed nuclei

DNA–DNA crosslinks Hincks et al. (1991); Reed et al. (1988)

Riddelliine (dehydroriddelliine)


DNA–protein crosslinksDNA–DNA crosslinks

Hincks et al. (1991); Kim et al. (1995, 1999)

Senecionine (dehydrosenecionine)



Coulombe et al. (1999); Hincks et al. (1991); Kim et al. (1995, 1999)

Seneciphylline (dehydroseneciphylline)



Hincks et al. (1991); Kim et al. (1995)

DNA strand breakHeliosupine In vitro alkaline elution Negative Hincks et al. (1991)Isatidin In vitro comet assay Positive Uhl et al. (2000)Jacobine In vivo alkaline elution Negative Petry et al. (1986)Latifoline In vitro alkaline elution Negative Hincks et al. (1991)Monocrotaline In vitro alkaline elution Negative Hincks et al. (1991); Petry et al. (1984)Monocrotaline In vitro comet assay Positive Silva-Neto et al. (2010)Retrorsine In vitro alkaline elution Negative Hincks et al. (1991)Riddelliine In vitro alkaline elution Negative Hincks et al. (1991)senecionine In vitro alkaline elution Negative Hincks et al. (1991)seneciphylline In vitro alkaline elution Negative Hincks et al. (1991)

Unscheduled DNA synthesisRetrorsine In rat hepatocytes following

in vivo treatmentPositive Griffi n and Segall (1986)

Riddelliine In rat and mouse hepatocytes following in vivo treatment

Positive Chan (1993); Chan et al. (1994); Mirsalis (1987); Mirsalis et al. (1993); NTP (2003)

Senecionine In rat hepatocytes following in vivo treatment

Positive Griffi n and Segall (1986)

Seneciphylline In rat hepatocytes following in vivo treatment

Positive Griffi n and Segall (1986)

Note: the pyrrolic PAs in brackets were also measured.18

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in a signifi cantly greater DHP-derived DNA adduct levels in the

endothelial cells than those in the parenchymal cells (Chou et al.,

2003c). Thus, the overall results suggest that DHP-derived DNA

adducts are, at least partially, responsible for the liver tumor

development (Fu et al., 2004). In addition, Fu et al. concluded that

this set of DHP-derived DNA adducts has the potential to be

utilized as a biomarker of PA exposure and tumorigenicity.

DNA cross-linking

PA metabolites contain two functional groups, at the C7 and the

C9 positions of the necine base (Fig. 1), that are capable of

binding to two sites in DNA or protein to form DNA or protein

cross-linking (Table 1). The antimitotic, toxic and carcinogenic

actions of PAs are thought to be caused, at least in part, by these

crosslinks (Coulombe et al., 1999; Hincks et al., 1991; Kim et al.,

1995, 1999).

Studies on the DNA–protein cross-linking activity of several

structurally diverse PAs were conducted using cells or isolated

nuclei. The pyrrolic PAs, dehydrosenecionine, dehydromonocro-

taline, dehydroseneciphylline and dehydroriddelliine readily

induced DNA–protein crosslinks, accounting for approximately

50% of the total cellular DNA crosslinks. The DNA–protein cross-

linking potency of PAs coincided with their known toxicity

potency in animals. Thus, it was concluded that DNA–protein

cross-linking was probably involved in PA-related toxicity

(Coulombe et al., 1999; Kim et al., 1995; Petry et al., 1984).

PA-induced DNA–DNA cross-linking was investigated in vivo

and in vitro using the model cross-linking PA monocrotaline.

Hepatic DNA damage induced by monocrotaline was evaluated

following i.p. administration to adult male Sprague–Dawley rats.

DNA–DNA crosslinks were characterized by the alkaline elution

technique; and a mixture of DNA–DNA interstrand cross-links and

DNA–protein cross-links was found (Petry et al., 1984). The active

metabolite of monocrotaline, dehydromonocrotaline, was also

demonstrated to mediate interstrand DNA cross-link formation

(Tepe and Williams, 1999). A study on the types of PA-induced

DNA–DNA cross-linking showed that the monocrotaline metabo-

lites produced piperidine- and heat-resistant multiple DNA cross-

links that were confi rmed by electrophoresis and electron

microscopy. It was proposed that the metabolites undergo rapid

polymerization to a structure capable of crosslinking several frag-

ments of DNA (Pereira et al., 1998). While one study found the

DNA–DNA cross-linking by dehydromonocrotaline lacked base

sequence preference (Rieben and Coulombe, 2004), another dem-

onstrated that the metabolites preferentially cross-linked DNA

duplexes containing the sequence 5’-CG (Weidner et al., 1990).

Coulombe et al. (1999) characterized the ability of diff erent

types of PAs to cross-link cellular DNA in cultured bovine kidney

epithelial cells. They found that every PA tested induced DNA

crosslinks. The relative potency of PAs in causing DNA cross-

linking, however, varied, with an order of seneciphylline > riddel-

liine > retrorsine > senecionine > heliosupine > monocrotaline >

latifoline > retronecine. Their studies suggest that PAs with a

macrocyclic necic acid ester and an α,β-unsaturated ester func-

tion are more potent cross-linkers. In addition, the stereochemi-

cal orientation of the ester linkage was found to have no eff ect

on biological activity (Hincks et al., 1991; Kim et al., 1999).

Unfortunately, the structures of the DNA crosslink adducts have

never been fully characterized; and the levels of their formation

have not been correlated with the tumorigenic potencies of

rodents treated with PAs. These warrant further investigation.

DNA strand breakage

Several reports have been published on PA-induced DNA strand

breakage (Table 1). Hincks et al. (1991) studied DNA crosslinks

and DNA strand breaks induced by several bifunctional PAs,

including seneciphylline, riddelliine, retrorsine, senecionine,

monocrotaline, heliosupine and latifoline, using the alkaline

elution assay. None of the PAs induced detectable amounts of

DNA single-strand breaks. Petry et al. (1984, 1986) confi rmed that

monocrotaline and Jacobine did not induced DNA single-strand

breaks using the alkaline elution assay. However, isatidine caused

a pronounced eff ect on human hepatoma cells (HepG2 cells)

using the single cell gel electrophoresis assay (Comet assay). The

lowest concentration that induced a signifi cant positive eff ect

was 500 μM (Uhl et al., 2000). The authors suggested that HepG2

cells catalyzed the activation reaction, leading to DNA strand

breaks. Glial cells from the human glioblastoma cell line GL-15

were treated with 1–5000 μM monocrotaline and the DNA strand

breaks were measured using the Comet assay (Silva-Neto et al.,

2010). The data showed that the treatment caused signifi cant

dose–response increases in cell DNA breaks. The confl icting

results from studies using the alkaline elution assay and Comet

assay have been explained by the greater sensitivity of the Comet

assay for detecting DNA strand breaks (Uhl et al., 2000).

Unscheduled DNA synthesis

The unscheduled DNA synthesis (UDS) test measures the DNA

repair synthesis after excision and removal of a stretch of DNA

containing the region of damage induced by chemical or physi-

cal agents. Therefore, this assay measures the repair of primary

DNA damage like DNA adducts. Several UDS studies have been

conducted to measure primary DNA damage induced by PAs

(Table 1). Riddelliine induced signifi cant elevations in UDS in rat

liver (Mirsalis, 1987; Mirsalis et al., 1993). UDS was also detected

in hepatocytes cultured from male and female rats and mice fol-

lowing 5 or 30 days of riddelliine treatment by gavage (Chan,

1993; Chan et al., 1994; NTP, 2003). Four PAs or PA metabolites,

senecionine, retrorsine, seneciphylline and 19-OH-senecionine,

along with four alkenals, trans-4-OH-2-hexenal, trans-4-OH-2-

nonenal, nonenal and hexenal, were measured in primary cul-

tures of rat hepatocytes using the UDS test. All eight compounds

exhibited positive, dose-related responses as measured by auto-

radiographic detection. The similar UDS responses by PAs and

alkenals suggest that trans-4-OH-2-hexenal is a toxic metabolite

of the PAs (Griffi n and Segall, 1986).

Chromosomal Damage Induced by PAs

Micronucleus assay

Micronucleus induction by PAs has been widely studied and the

results clearly demonstrate that PAs are strong clastogenic agents,

producing micronuclei in hepatocytes, bone marrow erythrocytes

and peripheral blood cells (Table 2). A PA mixture of crude integerri-

mine and retrorsine and pure integerrimine was extracted from

Senecio brasiliensis, which had been stored for more than 23 years

under variable conditions of temperature and humidity and exposed

to light. Both the mixture and pure PAs induced signifi cant increases

in micronucleus frequency in polychromatic erythrocytes (PCEs) of

mouse bone marrow (Santos-Mello et al., 2002). Monocrotaline and

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Table 2. Chromosome damage induced by pyrrolizidine alkaloids

Agent Testing system Results References

Micronucleus assayCrotalaria seeds In vivo peripheral blood of mice Positive MacGregor et al. (1990)Crude mixture of

integerrimine,

retrorsine and

impurities

In vivo polychromatic erythrocytes of mouse

bone marrow

Positive Santos-Mello et al. (2002)

Heliotrine In vivo polychromatic erythrocytes of mouse

adult bone marrow and fetal liver

Positive Sanderson and Clark (1993)

Integerrimine In vivo polychromatic erythrocytes of mouse

bone marrow

Positive Santos-Mello et al. (2002)

Isatidine in vitro rat hepatocytes Positive Muller-Tegethoff et al. (1997)Monocrotaline In vivo peripheral blood of mice Positive MacGregor et al. (1990)Monocrotaline In vivo polychromatic erythrocytes of mouse

adult bone marrow and fetal liver

Positive Sanderson and Clark (1993)

Monocrotaline In vitro rat hepatocyte micronucleus Positive Muller-Tegethoff et al. (1995, 1997)Riddelliine In vivo peripheral blood and bone marrow of

male mice

Week positive Chan et al. (1994)

Riddelliine In vivo peripheral blood of rats and mice Negative Chan et al. (1994); Mirsalis et al. (1993)Retrorsine In vitro rat hepatocyte micronucleus Positive Muller-Tegethoff et al. (1995, 1997)

Chromosome aberrationCrotalaria retusa Mouse bone marrow Positive Ribeiro et al. (1993)Fulvine Human blood cells of children suff ering from

veno-occlusive disease

Positive Martin et al. (1972)

Isatidine In vitro V79 cells with or without S9 Negative Muller et al. (1992)Isatidine In vitro V79 cells with primary hepatocytes Positive Muller et al. (1992)Heliotrine In vitro V79 cells with or without S9 Positive Takanashi et al. (1980)Heliotropium

curassavicum

In vitro CHO cells with or without S9 Positive Carballo et al. (1992)

Integerrimine Mouse bone marrow Positive Gimmler-Luz et al. (1990)Lasiocarpine In vitro V79 cells with or without S9 Positive Takanashi et al. (1980)Monocrotaline In vitro V79 cells with S9 or with primary

hepatocyte activation

Positive Muller et al. (1992)

Petasitenine In vitro V79 cells with or without S9 Positive Takanashi et al. (1980)Riddelliine In vitro CHO cells with S9. Positive Chan (1993); NTP (2003)Retrorsine In vitro V79 cells with or without S9; or with

primary hepatocytes

Positive Muller et al. (1992)

Senkirkine In vitro V79 cells with or without S9 Positive Takanashi et al. (1980)

Sister chromatid exchangedehydroretronecine In vitro human lymphocytes Positive Ord et al. (1985)Heliotrine In vitro V79 cells with primary chick embryo


Positive Bruggeman and van der Hoeven (1985)

Monocrotaline In vitro V79 cells with primary chick embryo



Riddelliine In vitro CHO cells with and without S9 Positive Chan (1993); NTP (2003)Seneciphylline In vitro V79 cells with primary chick embryo



Senkirkine In vitro V79 cells with primary chick embryo



Crotalaria seeds containing about 6.84% (dry weight) of monocro-

taline were administered to mice in their diet for 6 days; the treat-

ment increased the frequency of micronucleated cells in peripheral

blood (MacGregor et al., 1990). Clastogenic damage was evaluated

in mice following adult and transplacental exposure to heliotrine

and monocrotaline using the micronucleus assays of PCE in mouse

adult bone marrow and fetal liver, respectively. Both chemicals sig-

nifi cantly increased frequencies of micronucleated PCE in the adult

and fetal tissues, but heliotrine resulted in the larger increases in

mean value of micronucleated PCE. Also, the induction of micronu-

clei was signifi cantly higher in fetal than in adult cells. The induction

of micronuclei following heliotrine treatment showed a peak expres-

sion in PCE at 18 h after injection for adult bone marrow and at 24 h

for fetal liver (Sanderson and Clark, 1993). Isatidine, retrorsine and

monocrotaline were found to induce micronuclei in cultured rat

hepatocytes (Muller-Tegethoff et al., 1995, 1997).

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The frequency of micronucleated erythrocytes in mouse and

rat peripheral blood samples was not elevated after 4 or 13 weeks

of daily gavage treatments of riddelliine at doses up to 10 mg/kg

for rats and 25 mg/kg for mice. A weakly positive response was

noted in the peripheral blood and bone marrow of male mice

administered a single 150 mg/kg riddelliine by gavage (Chan,

1993; Mirsalis et al., 1993).

Chromosomal aberration

Many PAs induce chromosomal aberrations in mammalian cells

or in mouse bone marrow when they are appropriately metaboli-

cally activated (Table 2). Chromosomal aberrations were induced

by riddelliine in Chinese hamster ovary (CHO) cells only in the

presence of S9 (Chan, 1993; NTP, 2003). Heliotrine, lasiocarpine,

petasitenine and senkirkine induced chromosomal aberrations

in V79 cells (Takanashi et al., 1980). While heliotrine and petasit-

enine induced interchromosomal exchanges, lasiocarpine and

senkirkine caused chromatid gaps. Male and female C57Bl/6

mice were treated with integerrimine and their bone marrow

cells were collected for measurement of chromosomal aberra-

tions. The chromosomal aberrations were signifi cantly induced

in a dose responsive manner. The greatest frequency of chromo-

somal aberrations was detected 12 h after treatment (Gimmler-

Luz et al., 1990).

Muller et al. (1992) investigated chromosomal aberration

induction by monocrotaline, retrorsine and isatidine in diff erent

activation systems. Two hours of PA treatment of V79 cells with

S9 mix led to a strong and concentration-dependent increase for

retrorsine, but a negative response for isatidine (retrorsine

N-oxide) and a weak positive for monocrotaline. In contrast, an

18 h PA treatment of V79 cells in the presence of primary hepa-

tocytes resulted in clear concentration-dependent positive

responses for all three PAs. The authors postulated that primary

liver cells could reduce isatidine to retrorsine whereas the S9 mix

could not.

Two PA-containing plants were evaluated in the chromosomal

aberration test. Ribeiro et al. (1993) found that Crotalaria retusa

extracts containing monocrotaline induced chromosomal aber-

rations in mouse bone marrow cells. Heliotropium curassavicum

is a widely employed medicinal plant that contains PAs. In order

to analyze its genotoxic eff ects, Carballo et al. (1992) studied

chromosomal aberrations induced by Heliotropium extracts in

CHO cells with or without S9 mix. They found that the plant

extract induced chromosomal aberrations and the induction was

enhanced by the addition of an S9 fraction. The authors sug-

gested that the toxic eff ects were associated with PAs and their

N-oxides.

Genotoxicity studies on PAs in humans are rare. There is one

report on chromosomal aberrations in the blood cells of children

suff ering from veno-occlusive disease, believed to have been

caused by fulvine, a cyclic diester of retroneoine (Martin et al., 1972).

Sister chromatid exchanges

Sister chromatid exchange (SCE) is the exchange of genetic mate-

rial between two identical sister chromatids. SCE has been associ-

ated with chromosome damage and tumor induction. Several

PAs have been tested and found to be SCE inducers (Table 2).

Riddelliine induced SCEs in CHO cells with and without S9 (Chan,

1993; NTP, 2003). Heliotrine, monocrotaline, seneciphylline and

senkirkine were studied with the SCE assay in V79 cells. Treatment

of the cells with the PAs in the presence of co-cultured primary

chick embryo hepatocytes resulted in a strong induction of

SCEs. The rank order found was seneciphylline > senkirkine >

heliotrine > monocrotaline (Bruggeman and van der Hoeven,

1985). Dehydroretronecine, the common reactive pyrrolic metab-

olite of retronecine-type PAs, was measured in human lympho-

cytes for induction of SCE and produced a strong positive result

(Ord et al., 1985).

Mutations Induced by PAs

Mutations in bacteria

The Salmonella typhimurium/mammalian microsome test has

been extensively used for determining the mutagenicity of PAs

(Table 3). Retrorsine was measured using TA98, TA100, TA1535

and TA1537 tester strains in the presence of S9 and the PA was

mutagenic for TA1535 and TA1537, indicating that it induced

both basepair substitution and frameshift mutations (Wehner

et al., 1979). Dehydroretronecine also induced mutations in the

S. typhimurium base substitution strain TA92 (Ord et al., 1985). The

mutagenicities of clivorine, fukinotoxin, heliotrine, lasiocarpine,

ligularidine and senkirkine to S. typhimurium TA100 were demon-

strated by pre-incubation of the PAs with S9 mix and bacteria in

a liquid medium (Yamanaka et al., 1979). Riddelliine was muta-

genic in TA100 with, but not without, S9 activation. Riddelliine

was negative in strains TA97, TA98 and TA1535 (Chan, 1993). An

acetone extract of tansy ragwort produced positive mutagenic

responses in tester strains TA98, TA100, TA1535 and TA1537, with

S9 activation (White et al., 1984).

In spite of the results described above, there were contradic-

tory reports on the system for detection of mutagenicity of PAs.

Clark (1976) reported that PAs did not produce mutagenicity in

the S. typhimurium test system, even in the presence of a liver

microsome preparation. Rubiolo et al. (1992) found that several

PAs gave negative results in the test system. Retrorsine, seneciver-

nine, seneciphylline and extracts from PA-containing plants

Senecio inaequidens, S. fuchsia and S. cacliastershowed only

showed weak mutagenic activity. The authors concluded that the

S. typhimurium/mammalian microsome system was not a sensi-

tive assay for detection of PAs’ mutagenicity unless a suitable

activation enzyme system was applied.

Mutations in Drosophila

Drosophila test systems are particularly suitable for testing PAs

due to the fl y’s versatile metabolic bioactivation system and its

excellent sensitivity to cross-linking genotoxins (Candrian et al.,

1984). Heliotrine was a powerful mutagen in D. melanogaster

(Brink, 1969; Clark, 1959; Sivlingham and Brink, 1988). Sen-

eciphylline and senkirkine were found to produce sex-linked

recessive lethal in males of D. melanogaster using the 3-day

feeding method (Candrian et al., 1984). In addition, monocrota-

line was positive in Drosophila sex-linked recessive lethal assay

(Clark, 1976). Drosophila fl ies fed with milk from lactating rats

given an oral dose of 25 mg seneciphylline/kg showed 1.2% sex-

linked recessive lethal compared with 0.3% in controls (Candrian

et al., 1984).

Integerrimine was positive in the somatic mutation and recom-

bination wing spot test (SMART) in D. melanogaster. Analysis of

the dose–response data indicated that 85–90% of the genotoxic

events were due to mitotic recombination activity while 10–15%

of them resulted from somatic mutations (Campesato et al.,

1997).

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Table 3. Mutagenicity of pyrrolizidine alkaloids

Agent Testing system Results References

In bacteriaClivorine Salmonella typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Dyhydroretrosine S. typhimurium TA100 and T97 Positive Ord et al. (1985)Fukinotoxin S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Heliotrine S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Heliotrine Escherichia coli WP2 and WP2 uvrA Negative Green and Muriel

(1975)Isatidine S. typhimurium TA100 with S9 Negative Rubiolo et al. (1992)Lasiocarpine S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Ligularidine S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)LX201 S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Monocrotaline S. typhimurium TA100 with S9 Negative Rubiolo et al. (1992)Monocrotaline E. coli WP2 and WP2 uvrA Negative Green and Muriel

(1975)Retrorsine S. typhimurium TA1535 and TA1537

with S9Positive Wehner et al. (1979)

Retrorsine S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)Riddelliine S. typhimurium TA97, TA98 or TA1535

with or without S9Negative Chan (1993)

Riddelliine S. typhimurium TA100 with S9 Positive Chan (1993)Senecivernine S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)Senecionine S. typhimurium TA100 with S9 Negative Rubiolo et al. (1992)Seneciphylline S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)Senecio inaequidens and S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)S. fuchsia S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)S. cacliastershowed S. typhimurium TA100 with S9 Weakly positive Rubiolo et al. (1992)Senkirkine S. typhimurium TA100 with S9 Positive Yamanaka et al. (1979)Tansy ragwort S. typhimurium TA1535, TA1537, TA98

and TA100 with S9 Positive White et al. (1984)

In Drosophila7-Acetyl-intermedine Wing spot test Positive Frei et al. (1992)7-Acetyl-lycopsamine Wing spot test Positive Frei et al. (1992)Heliotrine Wing spot test Positive Brink (1969); Frei et al.

(1992); Sivlingham and Brink (1988).

Heliotrine Sex-linked recessive lethal test Positive Clark (1959)Indicine Wing spot test Positive Frei et al. (1992)indicine-N-oxide Wing spot test Positive Frei et al. (1992)Integerrimine Wing spot test Positive Campesato et al. (1997)Intermedine Wing spot test Positive Frei et al. (1992)Jacoline Wing spot test Positive Frei et al. (1992)Lycopsamine Wing spot test Positive Frei et al. (1992)Monocrotaline Wing spot test Positive Frei et al. (1992)Monocrotaline Sex-linked recessive lethal test Positive Clark (1959)Retrorsine Wing spot test Positive Frei et al. (1992)Senecionine Wing spot test Positive Frei et al. (1992)Seneciphylline Wing spot test Positive Frei et al. (1992)Seneciphylline Sex-linked recessive lethal test Positive Candrian et al. (1984)Senkirkine Wing spot test Positive Frei et al. (1992)Senkirkine Sex-linked recessive lethal test Positive Candrian et al. (1984)Supinine Wing spot test Negative Frei et al. (1992)Symlandine Wing spot test Positive Frei et al. (1992)Symphytine Wing spot test Positive Frei et al. (1992)

In rodentComfrey Transgenic rat cII assay Positive in liver and lung; The

major types of induced-mutations are G : C → T : A and tandem base substitutions

Mei et al. (2005); Mei and Chen (2007)

Riddelliine Transgenic rat cII assay Positive in liver and showed the liver endothelial specifi city; The major types of induced-mutations are G : C → T : A and tandem base substitutions

Mei et al. (2004a, b)

Riddelliine Mutations in the K-ras gene in mouse liver tumor induced by riddelliine

K-ras codon 12 G : C → T : A transversion; 58% incidence vs 0% in spontaneous tumors

Hong et al. (2003)

Mutations in the p53 gene in mouse liver tumor induced by riddelliine

75% incidence vs 0% in spontaneous tumors

Hong et al. (2003)

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Frei et al. (1992) examined 15 PAs and one PA N-oxide (indicine

N-oxide) for their genotoxic potency with respect to the struc-

ture/activity relationship in the wing spot test of D. melanogaster

following oral administration (Table 3). All PAs tested except the

C9-monoester supinine were clearly genotoxic. The results dem-

onstrated that the macrocyclic diester-type PAs were the most

genotoxic; the open diesters PAs were intermediate; and

7-hydroxy C9-monoester types of PAs were the least active.

Stereoisomeric PAs mostly showed similar activity. An increasing

number of hydroxy groups in the PA molecule seemed to reduce

genotoxic potency. A rank order with decreasing genotoxic

potency (senkirkine as 100%) follows: senkirkine (100.0), mono-

crotaline (90.0), seneciphylline (54.5), senecionine (39.1), 7-

acetyl-intermedine (22.5), heliotrine (13.4), retrorsine (8.3),

7-acetyl-lycopsamine (7.9), symphytine (3.8), Jacoline (1.8), sym-

landine (1.7), intermedine (0.49), indicine (0.27), lycopsamine

(0.19), indicine-N-oxide (0.07) and supinine (0.002).

Mutations in rodents

The mutagenicity of riddelliine in rat liver was investigated using

Big Blue transgenic rats (Mei et al., 2004a, b). Transgenic mutation

assays provide a unique opportunity for studying the induction

of tissue-specifi c mutation, and the assays also permit quantita-

tive measurements of mutant frequencies (MFs) in the PA-target

tissues and molecular analysis of the PA-specifi c mutational

spectra. Groups of six female transgenic Big Blue rats were

gavaged with 0.1, 0.3 and 1.0 mg riddelliine per kg body weight

5 days a week for 12 weeks and sacrifi ced 1 day after the last

treatment. The middle and high doses resulted in liver tumors in

an NTP bioassay (NTP, 2003). A signifi cant dose-dependent

increase in MF was found (Fig. 4).

Riddelliine mainly induces liver hemangiosarcomas that

develop from endothelial cells in rat or mouse liver (Chan et al.,

2003). To identify the cell type-specifi c response to mutagenicity

of PA, we separated the endothelial cells from the parenchymal

cells in the livers of Big Blue rats treated with riddelliine (Mei

et al., 2004a). While there was no diff erence in the cII MFs of liver

parenchymal cells in control and riddelliine-treated rats, the cII

MF of liver endothelial cells from treated rats was signifi cantly

greater than the cII MF of endothelial cells from control rats. The

induction of mutation also correlated with induction of DNA

adducts and tumors by riddelliine in rat liver (Fig. 4). These results

suggest that the relatively high mutagenicity of riddelliine in rat

liver endothelial cells may be partially responsible for the tumori-

genic specifi city of this agent.

Dose (mg/kg)

Mu

tan

t fr

eqeu

ncy

(x1

0–6 )

0

20

40

60

80

100

120

140

0 20 40 60 80 100

Neoplasm (%)

cII MF (×10-6)

DNA Adduct (×10-7)

HepatomaHemangiosarcoma

Frequency

1.21.00.80.60.40.20.0

Figure 4. Mutant frequencies induced by riddelliine in liver of female transgenic Big Blue rats. The upper panel shows mutant frequencies induced

by riddelliine treatment in the cII genes in rat liver as a function of dose. The lower panel compares mutant frequencies with other biological conse-

quences after riddelliine treatments in rat liver hepatocytes (solid bars) and endothelial cells (open bars). Data are from the literature (Chou et al., 2003c;

Hirono et al., 1978; Mei et al., 2004a).

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T. Chen et al.


A statistically signifi cant diff erence was found between the

mutational spectra from the riddelliine-treated and the control

rats. The major types of mutations induced by riddelliine were

G : C → T : A transversion and tandem base substitutions of GG →

TT and GG → AT (Table 4). The types of mutations induced by

riddelliine are consistent with riddelliine adducts involving G : C

base pairs (Chou et al., 2003a). Riddelliine reacts with guanine,

adenine and thymine, but not with cytosine, and the relative

reactivity of guanine with DHP is greater than other bases. The

GG → TT and GG → AT tandem base substitutions were believed

to result from intra-strand crosslinks in adjacent guanine bases

forming DHP-modifi ed dimers (Chou et al., 2003a; Mei et al.,

2004b). This unique spectrum may serve as a signature for

genetic damage produced by riddelliine and other PAs. The types

of mutations induced by riddelliine suggest that both mononu-

cleotide and dinucleotide DNA adducts involving G : C base pairs

are responsible for its mutagenicity (Mei et al., 2004a, b).

PA-containing comfrey is a rat liver toxin and carcinogen

(Hirono et al., 1978). In order to evaluate the mechanisms under-

lying its carcinogenicity, we examined the mutagenicity of

comfrey in the transgenic Big Blue rat model. Groups of six

6-week-old male Big Blue rats were fed either a basal diet or the

comfrey diet for 12 weeks. MFs were determined for the liver and

lung cII gene of the rats treated with comfrey. The MFs in both

liver and lung were increased by the comfrey treatment with a

much higher MF in liver compared to that in lung (Mei et al., 2005;

Mei and Chen, 2007). These results correlated with the previous

report that tumors were induced by comfrey in liver and liver was

the major target tissue (Hirono et al., 1978). Sequencing analysis

of the comfrey-induced cII mutant DNA showed the PA muta-

tional signature, with a high induction of G : C → T : A transver-

sions and tandem base substitutions (Table 4). Therefore, these

mutational data support the hypothesis that the mutations

induced by comfrey in rat liver and lung were due to the PAs in

comfrey (Mei et al., 2005; Mei and Chen, 2007).

Hong et al. (2003) examined the mutations occurring in the

K-ras protooncongene and p53 tumor suppressor gene in riddel-

liine-induced liver hemangiosarcomas. They found that 58%

riddelliine-induced tumors contained K-ras codon 12 G → T

mutation, a PA signature mutation, and 75% riddelliine-induced

tumors contained p53 mutations, the types of which were not

identifi ed. In contrast, spontaneous hemangiosarcomas from

control mice lacked mutations in both the K-ras and p53 genes.

It was concluded that p53 and K-ras mutations in riddelliine-

Table 4. Summary of the types of mutations in cII gene from riddelliine- or comfrey-treated and control Big Blue rats

Type of mutationControl (%) Treatment (%)

In liver In lung Riddelliine in liver Comfrey in liver Comfrey in lung

G : C → C : G 11 9 5 6 8

G : C → A : T 43 63 26 12 28

G : C → T : A 20 9 35 42 29

A : T → T : A 2 0 5 2 16

A : T → C : G 7 2 6 3 4

A : T → G : C 2 2 5 4 6

Frameshift 15 13 10 13 5Complex mutation 0 2 0 1 0Tandem–base substitution 0 0 8 17 4

Note: the data are from the literature (Mei et al., 2004b, 2005, 2007)

induced hemangiosarcomas most likely occurred as a result of

the mutagenic eff ects of riddelliine (Hong et al., 2003).

SUMMARY

There have been a number of outbreaks of human poisoning as

a result of ingestion of contaminated grain as well as case reports

of poisoning caused by intentional ingestion of herbal medicines

containing PAs. In recent years, there has been an increasing use

of herbal medicines and dietary supplements to treat various

chronic diseases and to promote health. Thus, humans may be

exposed to PAs via these herbs or dietary supplements, such as

Sympthytum spp. (comfrey), which have been deliberately

ingested. Although there are no epidemiological data regarding

the carcinogenicity of PAs in humans, a number of studies dem-

onstrated that various PAs are carcinogens in experimental

animals. Several PAs were classifi ed as possibly carcinogenic to

humans (group 2B) (IARC, 1976). Comparison of the total intake

resulting in human toxicity with the total doses to death observed

in the chronic toxicity studies in rats indicates that human beings

are more susceptible and suggests that humans may survive for

suffi cient time to develop cancer after only a brief exposure at

toxic levels or a longer exposure at a markedly lower levels (IPCS,

1988). Thus, a potential cancer risk for human beings should be

seriously considered. To assess the risk to humans, it is necessary

to determine whether tumorigenic PAs are mutagenic carcino-

gens because the selection of an appropriate model for conduct-

ing quantitative cancer risk assessment is based upon an

understanding of the chemical’s mode-of-action.

It seems that the carcinogenic activity of individual alkaloids

parallels their mutagenic behavior, but not their relative hepato-

toxicities (IPCS, 1988). Our review of the available information

demonstrated that the tumorigenic PAs are mutagenic. The

mutagenic activity is mediated by the formation of PA metabo-

lites binding to DNA, thus resulting in DNA damage, gene muta-

tions and chromosomal mutations. Since the mutagenicities

have been found in the tumorigenic target tissues and in onco-

genes of PA-induced tumors, it is reasonable to conclude that the

PAs induce tumors via a mutagenic mode of action. A postulated

mechanism of pyrrolizidine alkaloid carcinogenesis is shown in

Fig. 5.

PA metabolic activation plays a major role in their mutagenic-

ity. This is evidenced by the fact that the main genotoxic and

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tumorigenic target organ is the liver where the most PAs’ meta-

bolic activation occurs. The contradictory reports between in

vitro and in vivo mutagenicities of PAs also suggest the impor-

tance of PAs’ metabolism for their mutagenicities. PAs were not

mutagenic or showed weak mutagenic activity in S. typhimurium,

especially when suitable activation enzymes were not employed.

Surprisingly, no reports were found on mutagenicity of PAs using

in vitro mammalian cell assays, perhaps due to insensitivities of

these in vitro systems to PAs’ mutagenic insults. In contrast to the

in vitro systems, the in vivo test systems like Drosophila and trans-

genic rodents are particularly suitable for testing PAs due to the

in vivo system’s versatile metabolic bioactivation systems. The

mutagenic activities of some PAs that were identifi ed as negative

or weakly positive in bacteria were shown to be strong positives

in Drosophila or rodent test systems.

A large body of evidence shows that PAs induced DNA adducts

and DNA crosslinks while the results for PA-induced DNA breaks

were less clear. Pyrrolic esters or DHP can bind to DNA and gener-

ate DHP-derived DNA adducts. Two of the DNA adducts were

identifi ed as enantiomers of DHP-derived 7′-deoxyguanosin-

N2-yl adducts and the others were characterized as DHP-modifi ed

dinucleotides. The PA-induced DNA adducts could be formed in

common by all PAs. The observation that common types of muta-

tions, G : C → T : A and tandem base pair substitutions, were

induced by diff erent PAs strengthen this theory (Table 4). PAs

have proved to be strong cross-linking agents. The bifunctional

PAs are capable of forming DNA–DNA or DNA–protein crosslinks.

DNA–protein crosslinks comprise about half of the total cellular

DNA crosslinks. It also has been suggested that PA metabolites

can undergo polymerization to form a structure capable of pro-

ducing multiple DNA crosslinks. Diff erent PAs have diff erent

potencies in causing DNA cross-linking. PAs with a macrocyclic

necic acid ester and anα,β-unsaturated ester function demon-

strated the most potent cross-linking activity. While DNA adducts

may induce more gene mutations, DNA crosslinks tend to induce

chromosome mutations.

Pyrrolizidine alkaloids

Metabolic activation

PA metabolites (Pyrrolic ester, DHP, etc)

Conjugation (Glutathione, etc)

Detoxification Toxicity

Binding to protein, lipid, etc.

DNA adducts, DNA crosslinks, and DNA breaks

Binding to DNA

No repair (Apoptosis, etc.) DNA repair

Gene mutations (p53, ras, etc.) Chromosome mutations (LOH, etc.)

Error repair

Tumor initiation

Tumor promotion

Tumor progress

Tumor formation

Figure 5. Postulated mechanism of pyrrolizidine alkaloid carcinogenesis.

PAs are both gene mutagens and chromosomal mutagens,

perhaps more potent as chromosomal mutagens than as gene

mutagens. Many PAs were positive in micronucleus, chromo-

somal aberration and sister chromatid exchange assays which

detect chromosomal damage, and indicate the likely induction

of chromosomal mutations. Although many of the micronucleus

tests were conducted in surrogate tissues, bone marrow and

peripheral blood cells, PAs mainly were positive most of the time,

albeit weak. Chromosomal aberrations have been found in the

blood cells of children suff ering from veno-occlusive disease,

believed to be caused by the PA fulvine. PAs were positive in

many diff erent S. typhimurium strains, although the assay was

relatively insensitive to PAs’ mutagenicity. PAs induced mutations

in Drosophila, primarily measured with the wing spot or sex-

linked recessive lethal assays. The systems appeared to be par-

ticularly suitable for testing PA mutagenicity. The mutagenic

potency for a number of PAs has been ranked in these systems.

PAs induced mutations in the transgenic cII gene in rat liver and

lung. Riddelliine induced higher MFs in the endothelial cells than

the parenchymal cells in rat livers, which correlated with induc-

tion of hemangiosarcomas developed from rat endothelial cells.

Signature mutations of PAs have been identifi ed as G : C → T : A

transversion and tandem base substitutions. This signature

marker may be used as a fi ngerprint of PA exposure. Mutations

have been detected in the p53 tumor suppressor gene and K-ras

oncogene of liver tumors induced by riddelliine in mice. The

signature type of mutation, G : C → T : A, was found in the

oncogene.

The consistency between the induction of mutations and

tumors in liver suggests that gene and chromosomal mutations

are major factors in the induction of tumors by PAs. Gene and

chromosomal mutations in oncogenes and tumor suppressor

genes can initiate tumorigenesis. The initiated cells can be pro-

moted and progressed into tumors under the eff ects of muta-

tions induced by PAs in diff erent genes. Gene expression studies

have confi rmed that exposure of PAs signifi cantly changes genes

that are involved in cancer, cell death, tissue development, cel-

lular movement, tissue morphology, cell-to-cell signaling and

interaction, and cellular growth and proliferation. (Guo et al.,

2007; Mei et al., 2006, 2007). Although there is little evidence for

mutagenicity of PAs in humans, mutagenicity data from studies

in vitro, in vivo and in oncogenes provide strong evidence that

PAs are mutagenic carcinogens.

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