CURRENT AWARENESS DOCUMENT
PYRROLIZIDINE DERIVATIVE ALKYLATING AGENTS AND
RELATED PLANT ALKALOIDS:
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by:
David Y. Lai , Ph.D.
Yin-Tak Woo, Ph.D., D.A.B.T.
Science Applications Internation Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
SAIC Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc.
Extradi vi sional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5.3.2.3 Plant Alkaloids
Plant alkaloids comprise a large group of natural products which are
generally basic, nitrogenous heterocyclic compounds designated by the ending
"ine." Close to 4,000 of the structurally defined alkaloids are distributed
among 8-10% of the flowering plant species (34 out of 60 orders in the higher
plant system of Engler) were reported in 1978 (1). Many plant alkaloids are
toxic and display a wide spectrum of physiological activities; some of the
alkaloids discussed here are also carcinogenic and genotoxic. At one time,
colchicine and aristolochic acid were thought to be basic, nitrogenous hetero-
cyclics and were classified as alkaloids. The carcinogenicity of these two
^
compounds is covered in Section 5.3.2.6.4. Arecoline and other betel nut
alkaloids are discussed in Section 5.3.2.6.1.
A number of alkaloids also occur in animals, insects, algae, fungi and
bacteria (1). Ergot alkaloids, for instance, are present in the fungus,
Claviceps purpurea (see Section 5.3.1.4). Information on the toxicology of
alkaloids derived from organisms other than plants is still meager.
5.3.2.3.1 Pyrrolizidine Alkaloids
5.3.2.3.1.1 INTRODUCTION
Pyrrolizidine alkaloids constitute a large groups of compounds which
occur in plants of a wide botanical and geographical distribution. Close to
200 pyrrolizidine alkaloids have so far been found, distributed among more
than 350 plant species belonging to 12 families (principally Compositae,
Boraginaceae and Leguminosae) of the angiospermae. Many of these alkaloids
are highly hepatotoxic causing acute and chronic illness of grazing livestock
and farm animals in many parts of the world. Pyrrolizidine alkaloid poisoning
of humans has also occurred through the consumption of contaminated food
276
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grains. In Africa, Asia and other places, some pyrrolizidine alkaloid-
containing plants are used as food and folk medicines. Increased interest in
pyrrolizidine alkaloids has been stimulated by the findings that a number of
these naturally-occurring chemicals, when tested in long-term experiments in
animals, are tumorigenic. It is suspected that the high incidence of liver
cancer in some populations of the world may be related to the consumption of
pyrrolizidine alkaloid-containing plants.
In 1968, Bull, Culvenor and Dick (2) authored a comprehensive monograph
on the botanical distribution, chemistry, pathogenicity and other biological
properties of pyrrolizidine alkaloids. Subsequently, several periodic
i >
reviews, updating information on research in the field, particularly on the
toxicology, metabolism and carcinogenic action of the increasing number of
pyrrolizidine alkaloids have appeared (e.g., 3-10). Table XLIII lists the
names and uses of some plants in which carcinogenic pyrrolizidine alkaloids
have been found.
5.3.2.3.1.2 PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL EFFECTS
5.3.2.3.1.2.1 Physical and Chemical Properties. All alkaloids of this class
contain in their molecules the pyrrolizidine nucleus bearing a hydroxyl and a
hydroxymethyl group; this moiety is termed a necine. Necines form esters with
various Cc-Cjn branched-chain acids, called necic acids. Four types of
necines are recognized among the hepatotoxic and carcinogenic pyrrolizidine
alkaloids: (a) heliotridine, (b) retronecine, (c) isatinecine (N-oxide of
retronecine) and (d) otonecine. The necic acids are saturated or unsaturated,
hydroxylated or epoxidized mono- or di-carboxylic acids. Esters formed
between necines and necic acids can be classified into: (a) mono-esters, (b)
diesters and (c) macrocyclic diesters. The chemical structures of some pyr-
277
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c 2
Table XLIII
Names and Uses of Some Plants in Which Carcinogenic Pyrrolizidine Alkaloids Have Been Found3
Botanical name
Common name
Human use
Carcinogenic
pyrrolizidine alkaloid
Family Compos itae:
Senecio spp.
Tussilago farfara
Petasite japonicus
Farfugium japonicurn
Ligularla dentata
Family Boraginaceae:
Heliotropium spp.
Amsinckia intermedia
Ragwort, groundel,
stinking Willie,
Dan's cabbage, etc,
Coltsfoot
Coltsfoot
"Tsuwabuki"
(Japanese)
Common heliotrope,
caterpillar weed,
potato weed, etc.
Fireweed, tarweed,
fiddleneck, yellow
forget-me-not, etc.
Medicinal herbs in Africa,
Asia, Europe and Jamaica;
food in Japan
A cough medicine -in China,
Japan, and Europe^ food in
Japan
Food or a herbal remedy in
Japan
Food in Japan
Medicinal herbs in India,
Greece, and Eastern
Mediterranean, Africa and
South Africa
Retrorsine, isatidine,
riddelliine, senkirkine,
jacobine, seneciphylline
Senkirkine
Petasitenine, senkirkine
Petasitenine, senkirkine
Clivorine
Heliotrine, lasiocarpine
Lycopsamine, intermedine
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Table XL1IT (continued)
Botanical name
Common name
Human use
Carcinogenic
pyrrolizidine alkaloid
Symphytum officinale
Comfrey
Family Leguminosae
Crotalaria spp.
Rattle box, rattle
pad, wild lucerne,
earring plant, white
back, etc.
Green vegetable and tonic
in Japan; medicinal herb
in Europe and the United
States
Food and medicinal herbs in
India and Africa*/ "bush tea"
in West Indies
Symphyt ine
Retronecine, monocrotaline,
retrorsine, isatidine,
riddelliine, hydroxysen-
kirkine, seneciphylline
aSummarized from L.B. Bull, C.C.J. Culvenor and A.T. Dick [The Pyrrolizidine Alkaloids. Their Chemistry,
Pathogenicity and Other Biological Properties. Wiley, New York, 1968, 293 pp.], E.K. McLean [Pharmacol.
Rev. 22, 429 (1970)], IARC Monographs, Vol. 10 (1976) and Vol. 31 (1983), and I. Hirono, I. Ueno, S. Aiso,
T. Yamaji and M. Haga, Cancer Lett., 20, 191 (1983).
See Table XLIV for structural formulas and Table XLIX for carcinogenicity.
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rolizidine alkaloids which have been tested for carcinogenic activity are
shown in Table XLIV.
In pyrrolizidine alkaloids containing a ring oxo group (such as in
senkirkine, petasitenine and clivorine) the dotted lines represent fractional
valence bonds resulting from resonance between the limit structures:
,- -0
/0\ o8 ioi
- ^
')
CHo ^'3 CHj
Except for symphytine, which is an oil, the pyrrolizidine alkaloids dis-
cussed in this section are cr\staline colorless solids, mostly of low to
medium melting points. They are all optically active substances. In general,
the base strength and solubility in water and organic solvents of pyrrolizi-
dine alkaloids decrease in the order of: non-esters > mono-esters > diesters
> macrocyclic diesters. Hydrolysis and dehydrogenolysis of the ester groups
are the most important chemical reaction of these alkaloids. Also of
importance is the ready interconversion of the pyrrolizidine tertiary bases
and their N-oxides which are highly water soluble. Some physicochemical
properties of pyrrolizidine alkaloids are summarized in Table XLV.
5.3.2.3.1.2.2 Biological Effects Other Than Carcinogenicity
Toxic effects. Pyrrolizidine alkaloid-containing plants have long been
recognized as toxic to grazing animals and are responsible for many diseases
in farm stock (2). The prominent toxic effects in domestic animals are acute
and chronic liver lesions, lung damage, neurological symptoms and hemolytic
syndromes (3, 11). In experimental studies with rodents, the most frequently
affected organ is the liver. High doses of pyrrolizidine alkaloids cause
acute liver necrosis. Small doses produce chronic liver lesions characterized
278
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1 or 2
Table XLIV
Pyrrollzidine Alkaloids Which Have Been Tested for Carcinogenic Activity
H3C CH2OH
V'C
Isatidine
H .
C^/A / V
H- C/ ^ H dH C
0^Cx /°
Ov 0 CH2
H HsC\ f"3
\ ON CC
H,CX ^9* H OH r'
^ /
0^C\
0
CH3
Senkirkine R=-H Petasitenine
Hydroxysenkirkine R=-OH
o=c
s
°* °
CH3
Clivorine
RZ PHg-R|
R? CH2-R|
0
o
H
/c
OH
0=C
B
D
-------
-»f 2
Table XLIV (Continued)
Substituents to Fundamental Ring Structures A, B, C, & D
Heliotridine
Heliotrine
Lasiocarpine
Retrorsine
Monocrotal ine
Jacob ine
A
Rl R2
-OH -OH
CH(CH )
*l * J i
-0-C C CH-CH, -OH
II 1 1 3
0 OH OCH
HO-C-(CH-) CH3
-0-C C CH-CH. -0-C-C=CHCH,
II 1 1 3 II 3
0 OH OCH3 0
C
Ri Ro RT
-CH2OH -H =CH-CH3
CH-> H ( CH-i
1 -H
/°X
r*\\ 1] PU Pll
Vflln 11 v»n V>«»T
K J
B
Rl R2
Retronecine -OH -OH
CH(CH3)2
Lysopsamine -0-C C CH-CH. -Oil
nil3
0 OH OH
CH(CH-). CH
4 1 It 1
Symphytine -0-C C CH-CH, -0-C-C=CHCH,
Illl3 H 3
0 OH OH 0
D
Senec iphyll ine R = -CH3
Riddel 1 ine R = -CH2OH
*asymmetric carbon
-------
>f 2
Table XLV
Physicochemical Properties of Some Carcinogenic Pyrrolizidine Alkaloids3
Compound
Heliotrine0
Lasiocarpine0
Retronecine
Lycopsaraine
In termed ine
Symphyt ine
Retrorsine
M.p. (°C)
128
96.5-97
117-118
146.5-147
216-216.5
pKa
In 80% In
(fX]D (solvent) MCSd water
+17.6° (EtOH) 7.82 8.52
-3.0° (EtOH) 6.55 7.64
+49.6° (EtOH) 8.38 '
+3.3° (EtOH) 8.5
+4.7° (EtOH) 8.5
+3.65° (EtOH)
-18.0° (EtOH)
Solubility
2.64 g/dl; soluble in ethanol
0.68 g/dl; soluble in ethanol and
most non-polar solvents
Soluble in ethanol
Soluble in water and ethanol
Soluble in water and ethanol
Soluble in ethanol
Soluble in chloroform; slightly
Monocrotaline1
Jacobine
RiddelLiine
Senec iphy11ine
202-203
228
198
217
-62.0° (CHC13)
-15.0° (EtOH)
-55.0° (CHC13)
-46.3° (CHC13)
6.04
-109.5° (CHC13) 6.30
-139.0° (CHC13) 6.20 7.6
soluble in water and ethanol
1.21 g/dl; soluble in ethanol and
chloroform
Soluble in chloroform; sparingly
soluble in water, ethanol and
ether
Soluble in chloroform; slightly
soluble in water and ethanol
Soluble in chloroform; slightly
soluble in water and ethanol
-------
Table XLV (continued)
f 2
Compound
Isat idine
Senkirkine
M.p. (°C)
138
197-198
(<*}
-8.2
-12.
pKa
In 80% In
D (solvent) MCS water
0 (H20)
0° (EtOH)
Solubility
Soluble in water and
Soluble in chloroform
ethanol
and ethyl
Hydroxysenkirkine 124-125
Petasitenine 129-131
Clivorine 149-150
-2.0° (CHC13)
+5.3° (EtOH)
+63.8° (CHC13)
+80.2° (CHCU)
acetate; less soluble in water and
ethanol
Soluble in water, ethanol,
chloroform and hot acetone
Highly soluble in water
Soluble in chloroform
aSuramarized from L.B. Bull, C.C.J. Culvenor and A.T. Dick, "The Pyrrolizidine Alkaloids. Their Chemistry,
Pathogenicity and Other Biological properties." Wiley, New York, 1968, 293 pp.]; IARC Monograph Vol. 10,
International Agency for Research on Cancer, Lyon, France, 1976; T. Furuya, M. Hikichi and Y. litaka [Chem.
Pharm. Bull. 24, 1120 (1976)1; and K. Kuhara, H. Takanashi, I. Hirono, T. Furuya and Y. Asada [Cancer Lett.
JJ), 117 (19807T.
See Table XLIV for structural formulas.
cHalf-lives of alkaloids (in 0.5N NaOH at 25°C) in 1:1 aqueous ethanol: heliotrine = 8 days, lasiocarpine =
20 min., and monocrotaline = 18 min.; partition coefficients of alkaloids in oleyl alcohol/pH 7.3 buffer:
heliotrine = 0.11, lasiocarpine = 2.5, and monocrotaline = 0.082.
dMSC = methyl cellosolve.
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by the appearance of large parenchymal cells, "megalocytes." Some alkaloids,
especially mono-crot aline and seneciphylline, also cause lesions of the lung
(12). Liver necrosis and hepatic "veno-occlusive disease" are frequently seen
in humans ingesting pyrrolizidine alkaloid-containing plants as contaminants
of grain or as herbs for medicinal purposes (3, 13-16).
There is an inverse relationship between the acute hepatotoxicity and
both the water solubility and base strength of the pyrrolizidine alkaloids
(17). In order to be toxic, the compounds must contain the 1-hydroxymethyl-
pyrrolizidine structure, be unsaturated at the 1,2-position, and esterified at
one of the hydroxy groups. In general, macrocyclic diesters of retronecine,
i- N
such as retrorsine, are the most toxic alkaloids. Among the open esters,
diesters are more toxic than the monoesters; those of heliotridine are more so
than those of retronecine (12, 18). As compared with the corresponding
alkaloids, pyrrolizidine N-oxides are less acutely toxic when administered
parenterally but are similar in chronic effects (18). The acute toxicities
(LDcQ values) of some pyrrolizidine alkaloids in rats and mice are given in
Table XLVI.
Considerable evidence supports the conclusion that the hepatotoxic
effects of pyrrolizidine alkaloids are attributable to the pyrrolic metabo-
lites formed in the liver by enzymatic dehydrogenation (see Section
5.3.2.3.1.4). The enzyme system(s) involved has many characteristics of
mixed-function oxidases (20, 25). Factors such as species, age, sex, diet and
drug pretreatment, all of which influence the activity of mixed-function
oxidases, alter the hepatotoxicity of pyrrolizidine alkaloids in animals
(e.g., 26-28). Among livestock species, for instance, cattle and horses are
more susceptible than sheep and goat to pyrrolizidine alkaloid toxicity (2,
29). In laboratory animals, rats, mice and hamsters are more susceptible than
279
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Table XLVI
Acute Toxicity of Some Pyrrolizidine Alkaloids
Compound3
Retrorsine
Lasiocarpine
Monocrotaline
Isatidine
Heliotrine
Senkirkine
Seneciphylline
Jacob ine
Riddelliine
Lycopsamine +
intermediate
Symphytine
Species and route
Rat, i.p.
i .v .
Mouse, i.v.
i.p.
Hamster, i.p.
Rat, i.p.
i .v .
Mouse , i.v.
Hamster, i.v.
Rat , i.p.
i .v .
Mouse , oral
,i>v.
Rat , oral
i.p.
Mouse, i.v.
Rat, i.p.
i .v .
Mouse, i.v.
Rat , i.p.
Rat , i.p.
i .v .
Mouse , i .v .
Ra t , i.p.
Mouse, i.v.
Mouse, i.v.
Rat , i.p.
Mouse, i.p.
Rat , i.p.
LD50 (mg/kg)b
35 (M); 153 (F)
38
59
65 (M); 69 (F)
81
72 (M) , 79 (F)
88
85
68
95 (M) , 180 (F)
92
166
261
48
250
835
300 (M)
274
254
220
77 (M), 83 (F)
80
90
138 (F)
77
105
M.OOO (M)
300
130
Reference
(18,
(2)
(2)
(2)
(20)
(2)
(2)
(2)
(2)
(19)
(2)
(2)
(2)
(21)
(19,
(22)
(2)
(2)
(2)
(23)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(24)
(23)
19)
21)
aSee Table XLIV for structural formulas
Abbreviation: M = male, F = female.
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rabbits and guinea pigs (20, 30). In general, the males are more sensitive
than females and the newborns are more susceptible than older animals (26).
The protective effect of zinc on pyrrolizidine alkaloid-induced hepatotoxicity
in rats is primarily due to the blockage by the metal of the microsomal
conversion of the parent compounds to their toxic metabolites (28).
Acting either as mono- or bi-functional alkylating agents, the pyrrolic
metabolites of many pyrrolizidine alkaloids bind to, and inhibit, the
synthesis of cellular macromolecules (e.g., 17, 31-33). These metabolites are
also known to arrest mitosis of liver cells resulting in the development of
the chronic liver lesions, megalocytosis (31, 34).
? >
Mutagenic effects. The mutagenic and genotoxic activities of some car-
cinogenic pyrrolizidine alkaloids have been demonstrated in several assay
systems. The results of these studies are summarized in Table XLVII.
In microbial systems, preincubation of those alkaloids with S-9 mix is
required for the appearance of mutagenic activity. Yamanaka and coworkers
(35) noted that several pyrrolizidine alkaloids of the heliotridine (e.g.,
heliotrine and lasiocarpine) and otonecine (e.g., clivorine, petasitenine and
senkirkine) base types give positive mutagenic response in the Salmonella/
mammalian microsomal test. On the other hand, pyrrolizidine alkaloids of
retronecine base type (e.g., lycopsamine, raonocrotaline, retronecine and
seneciphylline) were not mutagenic under the same study conditions. These
observations led Yamanaka and associates to suggest that pyrrolizidine
alkaloid mutagenicity might be related to the necine base type of the com-
pounds. This view appears to be supported by findings of other investigators
that lasiocarpine (36) but not monocrotaline and jacobine (38), exhibit
mutagenic response in Ames strains of Salmonella typhimurium. Moreover,
280
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Table XLVII
Mutagenicity and Related Genotoxic Activities of Some Pyrrolizidine Alkaloids
Compound3
Heliotrine (H)
Lasiocarpine (H)
Senkirkine (0)
Petasitenine (0)
Clivorine (0)
Retrorsine (R)
Symphytine (R)
Monocrotaline (R)
Jacobine (R)
Seneciphylline (R)
Retronecine (R)
Lycopsamine (R)
Isatidine (I)
Salmonella Escherichia
typhimurium coli
+ (35) + (39)
+ (35, 36)
+ (35)
+ (35)
+ (35)
+ (37)
+ (9)
- (35, 38) + (39)
- (38)
- (35)
- (35)
- (35)
Aspergillus Drosophila V79 Chinese,
nidulans melanogasterc hamster cell
+ (40) + (41) + (44)
+ (40) + (41) + (44)
+ (42) + (44)
+ (44)
.
+ tt3)
+ (44)
+ (41)
+ (41)
+ (42)
+ (43)
DNA
repair
+ (45)
+ (45)
+ (45)
+ (45)
+ (46)
+ (45)
Chromosomal
aberrations
+ (44)
+ (44)
+ (44)
+ (44)
+ (47)
aLetters in parentheses are necine base types: H = heliotridine; 0 = otonecine; R = retronecine; I = isatinecine;
see Table XL1V for structural formulas.
In the presence of S-9 mix; "+" = positive; "-" = negative; numbers in parentheses are references.
cRelative mutagenic activity: lasiocarpine = 1.0; heliotrine = 0.9; monocrotaline = 1.6; jacobine =
8-Azaguanine-resistant mutation assay.
0.08.
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retrorsine (37) and symphytine (cited in ref. 10), both of retronecine base
type, induce mutation in these bacterial strains in the presence of S-9 mix.
The genotoxic DNA-damaging activity of monocrotaline and heliotrine could,
however, be shown in some repair-deficient strains of Escherichia coli which
proved to be more sensitive than Salmonella (39). Heliotrine and lasiocarpine
are also mutagenic in the fungus, Aspergilj.us nidulans (40).
Early studies with Drosophila melanogaster have indicated that monocrota-
line, lasiocarpine and heliotrine are potent mutagens; their N-oxides are less
active. Jacobine is only weakly mutagenic. The relative potencies of these
alkaloids with respect to mutagenicity in Drosophila are: lasiocarpine = 1.0;
t ^
heliotrine = 0.9; monocrotaline = 1.6; jacobine = 0.08 (41). More recently,
retrorsine, isatidine, senkirkine and seneciphylline have also been shown to
induce sex-linked recessive lethals in Drosophila (42, 43).
When tested in V79 cells derived from Chinese hamster lung, heliotrine,
lasiocarpine, petasitenine and senkirkine all induced chromosomal aberrations
and an 8-azaguanine-resistant mutation (44). Clastogenic activity was also
observed in Chinese hamster ovary cells following exposure to monocrotaline in
the presence of a microsomal activation system (47).
Wi 11 iams _et_ _al_. (45) have demonstrated the genotoxicity of lasiocarpine,
petasitenine, senkirkine, clivorine and monocrotaline in the hepatocyte
primary culture/DNA repair test. Retrorsine induces DNA repair replication in
livers of the rat (46).
A number of pyrrolizidine alkaloid-containing plants have also been
evaluated for the presence of mutagenic substances. With the addition of
liver microsomes from various mammalian species, an acetone extract of tansy
ragwort (Senecio jacobaea) produced positive mutagenic response in the tester
281
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strains of TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium (38). A
methanol extract of coltsfoot (Petasites japonicus) was also shown to be
mutagenic in the Salmonella strain, his G46, in an in vivo host-mediated assay
(48). Extracts of fresh leaves of the Russian comfrey, Symphyton officinale,
are mutagenic in the sex-linked recessive lethal test in Drosophila (49). The
mutagenic substance(s) in extracts of these plants remains to be investigated.
Teratogenic effects. Experimental data on teratogenic studies of pyr-
rolizidine alkaloids are surprisingly scanty. In the light of the alkylating
potential and antimitotic activity of their metabolites (see Section
5.3.2.3.1.4), it is likely that pyrrolizidine alkaloids might be terato-
genic. Green and Christie (50) have demonstrated a positive teratogenic
effect of heliotrine in the rat. Various skeletal malformations were observed
in offspring of rats receiving single doses (100 mg/kg) of heliotrine intra-
peritoneally during the second week of gestation. Other investigators have
shown that the administration of lasiocarpine to pregnant (51) or to lactating
(52) rats, in doses non-toxic to the dams, caused significant liver lesions in
the newborns and weanlings.
5.3.2.3.1.3 CARCINOGENICITY ANQ STRUCTURE-ACTIVITY RELATIONSHIPS
Overview. Pyrrolizidine alkaloids are among the first naturally occurr-
ing carcinogens found in products of plant origin. A report of tumor induc-
tion by pyrrolizidine alkaloids dates back to 1950 when Cook, Duffy and
Schoental (53) described the development of hepatomas in rats following
feeding with an alkaloidal fraction from tansy ragwort (Senecio jacobaea).
Since then, several other plants or plant extracts containing pyrrolizidine
alkaloids were shown to produce neoplasms in laboratory animals (rev. in 8,
54). The results of these studies are presented in Table XLVIII. While most
282
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Table XLVIII
Carcinogenicity of Some Pyrrolizidine Alkaloid-Containing Plants
Plant
Part
a
Care inogenic it y
Major
pyrrolizidine alkaloids
Reference
Senecio jacobaea
*
S. longilobus
S. cannabifol ius
Farfugium japonicum
Hel iotropium supinum
H. ramosissimum
Amsinckia intermedia
Petasites japonicus
Tussilago farfara
Symphytum offinale
Stem and leaf or
plant extract
Stem and leaf
Stem and leaf
Stem and leaf
Stem and leaf or
plant extract
Stem and leaf
Seeds
Flower stalk
Flower
Root and leaf
Liver tumors in rats
and chicks
Liver tumors in rats
Liver tumors in rats
Liver tumor in rats
Liver tumors in rats
Pancreas and kidney
tumors in rats
Seneciphylline , jacobine ,
senecionine, jaconine,
jacoline, jacodine
Seneciphylline
Brain tumors in rats
Kidney tumors in rats
Liver tumors in rats
and mice
Seneciphylline , retrorsine ,
riddelliineb
*
Seneciphylline , senecican-
nabine, jacozine
Senkirkine , petasitenine
Supinine, echinatine, helio-
supine, trachelanthyl-7-
angelylheliotridine, viri-
dofloryl-7-angelylhelio-
tridine
Heliotrine
Lycopsamine , intermedine
Petasitenine
Liver tumors in rats Senkirkine , senecionine
Liver tumors in rats
Symphytine , echimidine
(53, 55, 56)
(57)
(58)
(59)
(59)
(60, 61)
(62)
(61)
(54, 63-65)
(54, 66)
(54, 66)
aPlant parts were mixed and fed with the diet; plant extracts were injected intravenously.
Carcinogenic activity has been tested; see Table XLIX.
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of these plants (or their extracts) induce tumors in the liver of rats, dried
plants (stems and leaves) of the genus Heliqtropium elicit neoplasms of the
pancreas, the kidney (Ji. supinum) and the brain Ul. ramosissimum) in the rat
(60-62). Heliotropium supinum and JI. ramosissimum are medicinal herbs used in
East Africa (60, 62). Senecio cannabifolius, Petasites japonicus, Tussilago
farfara, Farfugium japonicum and Symphytum officinale are regarded as edible
plants in Japan (54, 59).
To date, only a few alkaloidal constituents of these plants have been
studied in long-term experiments for carcinogenic activity. However, all
hepatotoxic pyrrolizidine alkaloids and their metabolites, tested to date
i %
under adequate conditions have been found to be carcinogenic in the rat,
inducing tumors not only in the liver, but also in various other organs. The
carcinogenic pyrrolizidine alkaloids encompasses members of the monoester,
diester and macrocyclic diester categories (see Table XLIV for structural
formulas). The macrocyclic diester alkaloids appear to be more potent car-
cinogens than the monocyclic, open esters. Retronecine, which is not hepato-
toxic, is also carcinogenic when given to newborn rats. The carcinogenicity
studies of these compounds are summarized in Table XLIX. Retrorsine, mono-
crotaline, dehydroretronecine (85) and petasitenine (54) also exhibit positive
effects in in vitro cell transformation assays. Structure-activity relation-
ship analysis suggest that the double bond in the necine ring (1,2-dehydro-
pyrrolizidine) is essential for transforming cells in vitro (85).
Heliotrine. The possible carcinogenic effect of heliotrine, which occurs
in Heliotropium ramosissimum and several other species (2, 86), has been
investigated by Schoental (67) in male weanling Porton-Wistar rats. All
animals given one or two doses of heliotrine (300 mg/kg body weight) by
stomach tube died within 5 months. When the dose of heliotrine was reduced to
283
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p. i ot 2
Table XLIX
Carcinogenicity of Pyrrolizidine Alkaloids
Compound3
Heliotrine
Lasiocarpine
Dehydrohelio-
t rid ine
Retronecine
Lycopsamine ,
in termed ine
(mixture)
Symphytine
Retrorsine
Isat idine
Monoc rot aline
Dehydromono-
Species and strain
Rat ,
Rat ,
Rat,
Rat,
Rat ,
Rat ,
Rat ,
Rat ,
Rat ,
Rat ,
Rat ,
Rat,
Rat ,
Rat ,
Rat ,
Porton-Wistar
Fischer
Fischer 344
Fischer 344
hooded >
Wistar
Porton-Wistar
AC I
Wistar
Wistar
Wistar
Wistar
Sprague-Dawley
Spr ague-Da wley
Sprague-Dawley
Mouse , LAC A
Route
oral
i .p.
oral
oral
i.p.
s .c .
oral
i.p.
oral
or i.p.
oral
oral ,
i.p. or
topical
oral ,
i.p. or
topical
oral
s .c .
s .c .
topical
Principal organ
affected
Pancreas, liver,
urinary bladder
and testis
Liver , skin
Liver, hetnato-
poietic tissue
Liver, hemato-
poietic tissue
Multiple sites
CNS
Pancreas
Liver
Liver
Kidney
Liver
Liver, lung
Liver
Multiple sites
Pancreas
Skin
Reference
(67)
(6,
69)
(70,
(72)
(73)
(62)
(60)
(23)
(55,
75)
(61)
(55,
(76)
(77)
(78,
(80)
(81)
68,
71)
74,
74)
79)
crotaline
-------
Table XLIX (continued)
O- L
Compound8 Species and strain
Jacob ine, sene- Chick,
ciphylline
(mixture)
Riddelliine Rat, Wistar
Dehydroretro- Rat , Sprague-Dawley
nee ine
Mouse, Swiss
Mouse , LACA
Senkirkine Rat , ACI
Hydroxysenkir- Rat, Wistar
kine
Petasitenine Rat, ACI
(Fukinotoxin)
Clivorine Rat, ACI
Route
i.v.
oral
and i.p.
s .c.
s ,c .
and/or
topical
topical
i.p.
i.p.
oral
oral
Principal organ
affected Reference
Liver
Liver
Muscle
Skin
Skin
Liver
CNS
Liver
Liver
(56)
(74)
(78, 79)
(82)
(81)
(23)
(62)
(83)
(84)
aSee Table XLIV for structural formulas.
-------
230 mg/kg, one rat survived 27 months and developed adenomas of the pancreatic
islet cells. Co-administration of nicotinamide was proved to prevent liver
necrosis and suppress the toxicity of heliotrine. Six of 12 rats receiving
one or two intragastric doses of heliotrine (230 mg/kg) together with nicotin-
amide (350 mg/kg) by intraperitoneal injections, survived 22 months or
longer. Among the survivors, 3 had pancreatic islet cell tumors, accompanied
in one of them by a hepatoma and in another by tumors of the urinary bladder
and testis. Such tumors were not found in control rats.
Lasiocarpine. The carcinogenicity of lasiocarpine has been repeatedly
demonstrated in Fischer rats by Reddy and his coworkers (68-71). In one
i >
experiment, lasiocarpine was administered intraperitoneally to 25 male rats at
doses of 7.8 mg/kg body weight, twice weekly for 4 weeks and then once weekly
for 52 weeks. Of the animals surviving after the treatment period, 61%
(11/18) developed hepatocellular carcinomas, 33% (6/18) developed squamous-
cell carcinomas of the skin, and 28% (5/18) had pulmonary adenoma (68, 69).
Both the carcinomas of the liver (68) and of the skin (69) were transplant-
able. At doses of 0.39, 0.78 and 1.56 mg/kg, lasiocarpine also induced low
incidences of liver neoplasms in male and female rats by repeated i.p. injec-
tions (6). When lasiocarpine was fed to 20 male rats in the diet (50 ppm) for
55 weeks, malignant tumors were found in 17 animals between 48 and 59 weeks:
9 developed angiosarcomas of the liver (1 of these also had carcinomas of the
skin), 7 developed hepatocellular carcinomas, and one developed lymphomas.
The angiosarcoma from one rat was successfully transplanted through 4 genera-
tions (70). Administration of thioacetamide, a liver carcinogen which
stimulates cell proliferation (see section 5.2.2.6, Vol. IIIB) stimulates the
development of hyper plastic nodules and carcinomas of the liver of rats
treated with lasiocarpine (71, 87).
284
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Under the bioassay conditions of the U.S. National Cancer Institute (72),
lasiocarpine is also carcinogenic in Fischer 344 rats, producing angiosarcomas
arid hepatocellular tumors in both sexes and hematopoietic tumors in female
animals. Statistically significant incidences of these neoplasms were
observed when groups of 24 rats of each sex were administered lasiocarpine in
the diet at doses of 7, 15 or 30 ppm for 104 weeks.
Dehydroheliotridine. Dehydroheliotridine is a major metabolite of helio-
tridine-base alkaloids such as heliotrine and lasiocarpine (see Section
5.3.2.3.1.4 on Metabolism and Mechanism of Action). When a group of 24 hooded
strain rats was injected with dehydroheliotridine intraperitoneally once every
> ^
4 weeks for 32 weeks (first dose, 76.5 rag/kg; second dose, 65 mg/kg; remainder
of doses, 60 mg/kg), 11 malignant and benign tumors emerged in 6 animals. In
addition to one cystic cholangioma in the liver, other neoplasms occured in
the pancreas, lung, adrenal gland, forebrain and the gasotrintestinal tract.
This wide spectrum of tumors indicates that the tissue targets of dehydro-
heliotridine are predominantly extrahepatic. Co-administration of thioacet-
amide enhances the hepatic toxicity but not the carcinogenicity of dehydro-
heliotridine (73).
Retronecine. Retronecine is the only non-hepatotoxic and non-ester ifled
pyrrolizidine alkaloid that is carcinogenic. In a small-scale study, a spinal
cord tumor was noted in one of 10 male Wistar rats 201 days after a single
s.c. dose (600 mg/kg) of retronecine, administered when the rats were new-
born. Such tumors were not found in hundreds of historical control rats.
Among 6 female newborn rats given a single s.c. injection of 1,000 mg/kg
retronecine, 5 developed pituitary tumors and one had a mammary tumor (62).
285
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Lycopsamine and Internedine. These two alkaloids are stereoisomers which
may be isolated from tarweed (Amsinckia intermedia), a plant known to cause
severe livestock losses from liver injuries in the United States. One adenoma
and one adenocarcinoma of the islet cells and one adenoma of the exocrine
pancreas were observed in 3 of 15 male Wistar rats given single doses (500-
1,500 mg/kg) of a mixture of lycopsamine and intermedine by stomach tube.
Pancreatic tumors of these types were considered to be very rare in these
animals (60) .
Symphytine. The carcinogenicity of Russian comfrey (Symphytum
officinale) is attributed to symphytine. Among 20 male ACI rats which
i >
received i.p. injections of symphytine at the dose of 13 mg/kg body weight
(10% of the LDCQ), twice weekly for 4 weeks and then once a week for 52 weeks,
one had a liver cell adenoma and 3 developed hemangioendothelial sarcomas in
the liver. These tumors were similar to the ones observed in rats fed diets
containing roots and leaves of Russian comfrey (88).
Retrorsine. The chronic liver lesions produced in rats by this highly
toxic alkaloid were noted by Schoental et al. (55) in early 1954. Fourteen
Wistar rats (10 male and 4 female) which were administered 0.03-0.05 mg/ral
retrorsine in the drinking water 3 days a week, survived from 10 to 24
months. On examination, nodular or microscopic foci of hyperplasia of the
liver were found in 9 of the 10 male rats. The nodules in 4 of these rats
were histologically identified as hepatomas. Subsequent studies, in which
weanling Wistar rats were given repeated i.p. doses (74) or single large doses
(30 mg/kg) of retrorsine by stomach tube (75), also showed significant inci-
dences of tumors. In addition to hepatomas, other neoplasms observed in the
retrorsine-treated rats were tumors of the lung (55, 75), mammary gland,
spleen, uterus (75) and kidney (61).
286
-------
Isatidine (Retrorsine-N-oxide). The N-oxide of retrorsine seems to be
even more hepatocarcinogenic than its parent compound. Of 29 Wistar rats of
both sexes receiving 0.03-0.05 mg/ml of isatidine in the drinking water, 13
developed neoplasms and hyperplastic nodules of the liver; the tumors in one
of the rats metastasized. Although no tumors were found in the liver, 8 other
rats treated with isatidine had preneoplastic nodules. Supplements with
choline did not protect the liver from the carcinogenic action of isatidine
(55). Liver tumors are also induced in the rat when isatidine is administered
by painting on the neck (55) or by i.p. injection (74).
Monocrotaline. Although monocrotaline has long been implicated as a car-
f %
cinogen, conclusive data have not been reported until recently. In 1955,
Schoental and Head (76) noted lesions of the liver and lung indicative of neo-
plasia in Wistar rats exposed chronically to monocrotaline. Newberne and
Rogers (77) reported later that hepatocellular carcinomas were produced in 31%
(24/77) of Sprague-Dawley rats receiving repeated doses (25 mg/kg for 4 weeks
then 8 mg/kg for 38 weeks) of the alkaloid by stomach tube. Allen and
coworkers (78, 79) administered monocrotaline (5 mg/kg body weight) biweekly,
by s.c. injection to a group of 60 male Sprague-Dawley rats for a year.
Twelve months after the treatment, 10 animals had pulmonary adenocarcinomas, 5
had well-differentiated hepatocellular carcinomas and 4 developed rhabdomyo-
sarcomas. Additionally, 8 adrenal adenomas, 3 acute myelogenous leukemias and
one renal adenoma were observed in the treated rats. A high incidence (70%)
of insuloma of the pancreas also occurred in 23 male Sprague-Dawley rats 500
days after a single subcutaneous injection of 40 mg/kg monocrotaline (80).
Dehydromonocrotaline. The carcinogenic potential of dehydromonocrota-
line, a putative primary metabolite of monocrotaline (see Section 5.3.2.3.1.4
on Metabolism and Meghanism of Action) has been assayed by mouse skin-
287
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painting. Repeated topical doses (2.5^nnol) of dehydromonocrotaline, followed
by repeated treatment with croton oil, resulted in the development of malig-
nant tumors of the skin in 5 of 10 LACA strain mice (81). Without the tumori-
genesis promoter, no carcinogenic effect was observed (81, 89).
Jacobine and Seneciphylline. A mixture of jacobine and seneciphylline
(primarily seneciphylline), purified from ragwort (Senecio jacobaea), was
tested for carcinogenic activity in the chick (56). Primary liver tumors
occurred in 6 of 24 chicks receiving weekly i.v. doses (20-35 mg/kg) of the
alkaloid mixture for up to 8 weeks or until death.
The chronic changes in,tjie liver of rats caused by seneciphylline are
closely similar to those in rats given lasiocarpine or other carcinogenic
pyrrolizidine alkaloids. Liver hyperplastic nodules developed in Wistar rats
after a single oral dose (40 mg/kg) of seneciphylline (90). Plants containing
seneciphylline and jacobine have been shown to produce liver tumors in the rat
(see Table XLVII).
Riddelliine. Riddelliine occurs in the carcinogenic plant Senecio
longilobus, as well as in several other Senecio species (2). Chronic admini-
stration of this alkaloid to rats produced liver lesions characteristic of
many hepatocarcinogenic alkaloids of this class. In a chronic study in which
20 male and female Wistar rats were given riddelliine in their drinking water
(0.02 mg/kg) and by i.p. injections (25 mg/kg), 9 animals had hyperplastic
nodules of the liver and one had a hepatic sarcoma. No such lesions were
found in 15 controls (74).
Dehydroretronecine. Dehydroretronecine is the secondary metabolite of
monocrotaline and perhaps of other retronecine-based alkaloids (see Section
5.3.2.3.1.4 on Metabolism and Mechanism of Action). The direct-acting car-
cinogenic effects of this metabolite have been studied in both rats and mice.
288
-------
Of 60 male Sprague-Dawley rats receiving biweekly s.c. injections of
dehydroretronecine (20 mg/kg for 4 months, then 10 mg/kg for 8 months), 39
developed rhabdomyosarcomas at the injection site. Metastasis occurred in 5
cases (78, 79). Significant incidences of basal cell and squamous cell car-
cinomas of the skin were found in 92 Swiss mice given repeated s.c. injections
and/or topical applications of dehydroretronecine (82). Repeated topical
doses (5 iimol/dose) of dehydroretronecine also induced skin tumors, in mice of
the BALB/c and LACA strains (81, 89). Similar carcinogenicity toward the skin
of LACA strain mice were shown by the synthetic compounds, 2,3-bistrimethyl-
acetoxymethyl-1-raethylpyrrole and 2,3-bis-hydroxyraethyl-l-methylpyrrole but
not by 2,3-bis-hdyroxymethyl-5-methyl-l-phenylpyrrole (see Table L).
Senkirkine. The active compound in coltsfoot (Tussij.ago farfara) is sen-
kirkine. When a group of 20 ACI strain rats were given i.p. injections of
senkirkine (22 mg/kg) freshly prepared from the flowers of the plant, twice
weekly for 4 weeks and then once a week for 52 weeks, 9 animals developed
liver cell adenomas similar to those observed in rats fed diets containing the
flowers of coltsfoot (23).
Hydroxysenkirkine. Schoental and Scavanagh (62) reported that a single
i.p. dose of hydroxysenkirkine (300 mg/kg), isolated from an East African
plant Crotalaria laburnifolia (a variety of laburnifolia) , induced an astro-
cytoma of the cerebrum in one of 4 male rats of the Wistar-Porton strain.
Tumors of this type were not seen among hundreds of historical controls.
Petasitenine (Fukinotoxin). There is evidence indicating that petasi-
tenine may be responsible for the carcinogenic activity of Petasites
japonicus, a variety of coltsfoot (83). The same types of tumors (i.e.,
hemangioendothelial sarcomas and liver cell adenomas) were found in 8/10 ACI
289
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Relative Carcinogenic
Compound
Table L
Activity of Dehydroretronecine and Structurally-Related Compounds
Toward the Skin of LACA Mice
Structure
Dose
(uraol/mouse) incidence
Dehydroretronec
HO
5.0
25%
2,3-Bis-trimethylacetoxy-
methyl-1-methyl pyrrole
(CH3)3C
C-O
O
II
0-C-C(CH3).
0.5
90%
2,3-Bis-hydroxymethyl-l-
methylpyrrole
5.0
25%
2,3-Bis-hydroxymethyl-5-
methyl-1-phenylpyrrole
5.0
9%c
Modified from: A.R. Mattocks and J.R.P. Cabral [Cancer Lett. 17. 61 (1982)
Applied to the shaved backs of mice at weekly intervals for up to 47 weeks.
cStatistically not significant as compared with control incidence.
-------
strain rats receiving a 0.01% solution of petasitenine in the drinking water,
as in the rats fed a diet containing the flower stalks of Petasites japonicus
(54, 63, 64).
Clivorine. Like senkirkine, hydroxysenkirkine and petasitenine,
clivorine is a pyrrolizidine alkaloid of the macrocyclic diester type con-
taining otonecine as the necine base. Among 12 ACI strain rats ingesting a
0.005% solution of clivorine in drinking water for 340 days, 2 developed
hemangioendothelial sarcomas and 6 had neoplastic nodules of the liver. The
hemangioendothelial sarcoma in one rat showed metastasis in the lung. No
liver tumors or nodules were found in the controls (84).
i ^
5.3.2.3.1.4 METABOLISM AND MECHANISM OF ACTION
There is considerable evidence that the toxicological effects of pyrro-
lizidine alkaloids are not due to the compounds themselves but to metabolites
formed in the liver. Much of the metabolic studies of these toxic alkaloids
have been carried out in the laboratory of Mattocks, White and associates in
Great Britain (20, 91-94). The general scheme for the metabolism of some car-
cinogenic pyrrolizidine alkaloids is shown in Fig. 11.
Pyrrolizidine alkaloids which are esters of heliotridine (e.g., helio-
trine, lasiocarpine) or retronecine (e.g., lycopsamine, symphytine, retror-
sine, monocrotaline, jacobine, riddelliine and seneciphylline) undergo
N-oxidation to yield N-oxide derivatives, and C-hydroxylation to give dehydro-
alkaloids (primary pyrrolic derivatives) in the liver of the rat. In vitro
studies have shown that both reactions are catalysed by typical mixed-function
oxidases in the microsomes (91). The formation of N-oxides, which are more
water soluble and have low toxicity, is considered to be a detoxifying reac-
tion. The toxic effects observed following administration of N-oxide
290
-------
CH,OR
N-oxide
derivatives
Esters of
Heliotridineor
Retronecine
CHOR
Dehydroalkaloids
(Pyrrolic derivatives)
-HoO
Esters of otonecine
HO Cri>OH
\*
CHoNu
Reaction products
with nucleophile
Fig. 11. General scheme for the bioactivation of some pyrrolizidine
alkaloids. Asterisks (*) denote asymmetric carbon atoms; Nu denotes nucleophiK
-------
alkaloids depend on the reduction back to their parent compounds a reaction
which has been shown to occur in the gut of the rat (21) and in the rumen of
sheep (2). Both the C-7 and C-9 of the dehydroalkaloids are highly electro-
philic, especially when they bear an acyloxy grouping. However, under
slightly acid conditions, the C-7 and C-9 dehydroalkaloids are also strongly
electrophilic when they bear a hydroxyl group. The dehydroalkaloids thus are
potentially bi- or mono-functional alkylating agents which may readily react
with nucleophilic constituents in the cell or may react with water to yield
more stable dehydroaminoalcohols, such as dehydroheliotridine and dehydro-
retronecine (secondary pyrrolic derivatives).
i ^
Esters of otonecine (e.g., senkirkine, hydroxysenkirkine, petasitenine
and clivorine) also yield reactive pyrrolic metabolites by initial N-de-
methylation, to give an 8-hydroxypyrrolizidine, and then the dehydroalkaloid
of the corresponding ester.
Strong evidence has pointed to the pyrrolic metabolites as the major
metabolites involved in the hepatotoxic, mutagenic and carcinogenic activities
of pyrrolizidine alkaloids. However, it is not known whether either or both
the dehydroalkaloids and the dehydroaminoalcohols are proximate carcinogens.
Black and Jago (95) demonstrated that dehydroheliotridine, the major pyrrolic
metabolite of heliotrine and lasiocarpine, can interact with calf thymus DNA
in vitro. Alkylation of DNA by dehydroretronecine has also been shown in
vitro (32, 96-98) and in vivo (32, 99). Chemical and spectral analyses have
revealed that the major reaction product of dehydroretronecine with deoxy-
guanosine (dGuo) is 7-(deoxyguanosine-N -yDdehydroretronecine, indicating
that the reactive electrophile derived from the protonated dehydroretronecine
readily alkylates deoxyguanosine (dGuo) at the N position (98).
291
-------
CH2OH dGuo CH2OH
dGuo
Dehydroretronecine deoxyguanosine adduct
Following i.p. administration of monocrotaline to Sprague-Dawley rats,
DNA-DNA interstrand cross-links and DNA-protein cross-links are formed in the
liver cells. These DNA cross-links have been suggested to be instrumental in
the hepatocarcinogenicity of monocrotaline (99).
An alternative hypothesis is that epoxide derivatives may be the hepato-
toxic and carcinogenic metabolites of pyrrolizidine alkaloids (5, 100). That
the open diester alkaloids are less toxic and carcinogenic than the macro-
cyclic esters may be explained by steric hindrance of the epoxidation of the
i >
C1-C2 double bond by the ester side chain at C-l in the open ester alkaloids
(100).
Recent research shows that jacobine, the pyrrolic derivative of dehydro-
retronecine, and isobutyryl retronecine, but not retronecine nor the pyrroLic
derivative of isobutyryl dehydroretronecine pyrrole, induce gene expression of
endogenous avian tumor virus in cultured chick embryo fibroblasts (101).
5.3.2.3.1.5 ENVIROIMENTAL SIGNIFICANCE
Plants containing pyrrolizidine alkaloids are so abundant and widespread
that they are found in almost every region of the world. As many as 6,000
species, or 3Z of the world's flowering plants are estimated to contain some
levels of pyrrolizidine alkaloids (cited in ref. 86). For example, among the
24 species of Heliotropium, currently collected along the border of Mexico and
the United States, all contain various amounts of unsaturated pyrrolizidine
alkaloids (102). The number of species listed in a recent compilation (86)
are believed to be only a small proportion of the pyrrolizidine alkaloid-
containing plants which actually exist world-wide. According to Smith and
292
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Culvenor (86), all species in the family of Boraginaceae and the genera
Senecio, Crotalaria and Eupatorium should be regarded as potentially hepato-
toxic .
Sporadic outbreaks of diseases and death in agricultural livestock and
farm animals due to consumption of grass or hay contaminated with pyrrolizi-
dine alkaloid-containing plants have occurred in many parts of the world.
Several species, in particular those belonging to the genera of Senecio,
Crotalaria and Heliotopium, are classic poisonous plants. Senecio jacobaea
(Tansy ragwort or Stinking Willie), for instance, is a common contaminant in
pastures and is responsible for the "Pictou disease" of cattle and horses in
f >
Canada and the "Winton disease" of livestock in New Zealand (2). Livestock
poisoning by consumption of S. jacobaea and other pyrrolizidine alkaloid-
containing plants in Australia and the Pacific Northwest of the United States
has been a serious problem of considerable economic importance (11).
Human exposure to pyrrolizidine alkaloids may occur through the consump-
tion of plant materials contaminating cereal grains. For example, from 1935
to the mid-1950"s, an epidemic of poisoning took place in the U.S.S.R. because
of the contamination of bread made from wheat, barley or millet containing
Heliotropium lasiocarpum (see 16). During 1974-1975, an outbreak of
veno-occlusive disease in northwestern Afghanistan was identified to be due to
the consumption of flour made from wheat contaminated with seeds of
Heliotropium popovii subsp. gillianum, which contains heliotrine and lasio-
carpine (13, 103). Another outbreak of veno-occlusive disease in central
India between 1975 and 1976 has been correlated with the ingest ion of cereals
mixed with seeds of a plant, of the Crotalaria species, containing hepatotoxic
pyrrolizidine alkaloids (14). Many incidences of poisoning through consump-
tion of food, contaminated with plants of the genus Senecio, were reported in
South Africa (see 3, 16).
293
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Exposure of humans to these alkaloids also occurs as a result of the use
of pyrrolizidine alkaloid-containing plants as medicinal herbs. In partic-
ular, many species in the genera of Senecio, Heliotropium and Crotalaria are
used in countries of Asia, Africa and Europe as herbal remedies for the treat-
ment of a wide range of ailments (see Table XLIII). Cases of human poisoning
due to the consumption of these plants or plant extracts for medicinal
purposes have been recorded (see 11,16).
In addition, humans may be exposed to pyrrolizidine alkaloids through
food products since several pyrrolizidine alkaloid-containing plants such as
coltsfoot (Petasite japonicus) or comfrey (Symphytum officinale) have been
used in Japan, Australia, Europe and the United States as vegetables and for
the preparation of herb teas (see Table XLIII). Furthermore, small amounts of
pyrrolizidine alkaloids have been detected in the milk of tansy-fed cows (104)
as well as in honey produced from the nectar of Senecio jacobaea (105) and
-^
EChiurn plantagineum (106).
So far, there is no epidemiologic evidence that links pyrrolizidine
alkaloids with carcinogenesis in humans. However, the high incidence of liver
cancer in the Bantu population of South Africa has been related to the use of
Senecio plants for medicinal and other purposes (18). The desert Bedouins in
Kuwait, who use Heliotropium ramosissimum (which contains heliotrine) as an
herbal remedy and for certain other purposes, have a higher incidence of liver
cancer than do town dwellers (15).
5.3.2.3.2 Plant Alkaloids Other Than Pyrrolizidine
5.3.2.3.2.1 INTRODUCTION
In addition to pyrrolizidine alkaloids, several other plant alkaloids
have received considerable attention because of their therapeutic and pharraa-
294
-------
cologic actions. The medicinal use of plants containing reserpine, sanguin-
arine, emetine and quinine in various parts of the world dates back almost to
antiquity. Despite the advent of synthetic drugs, reserpine and quinine are
still prescribed presently for the treatment of hypertension and malaria,
respectively. Recent research has discovered that vinblastine, vincristine,
acronycine and emetine are effective against certain neoplasms and are valu-
able agents in cancer chemotherapy. Nicotine and caffeine have no important
clinical application; however, their pharmacological actions have been well
established. Experimental studies indicate that several of these plant alka-
loids are genotoxic and teratogenic. The possibility that they may produce
' ^
neoplasms in animals and humans has also been investigated. A number of
general reviews on these alkaloids has appeared (1, 107-114).
5.3.2.3.2.2 PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL EFFECTS
5.3.2.3.2.2.1 Physical and Chemical Properties. The structural formulas of
some plant alkaloids tested for carcinogenic activity are shown in Table LI.
Like many other alkaloids, they contain nitrogen atom(s) within a heterocyclic
ring system. Except for caffeine, which is a xanthane derivative, others are
biosynthesized from amino acids and are basic. Nicotine is one of the few
liquid alkaloids; others are colorless crystalline solids with well defined
melting points. They are highly susceptible to decomposition by heat or
light; however, salification with inorganic acids increases their stability.
Some important physicochemical properties of these alkaloids are given in
Table LII.
5.3.2.3.2.2.2 Biological Effects Qther Than Carcinogenicity
Pharmacological effects. For decades, many of these alkaloids received
much more attention from pharmacologists than from toxicologists. The phar-
295
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.1 of 2
Table LI
Sane Plant Alkaloids Which Have Been Tested for Carcinogenic Activity
CH30
H.COOC
OCH
Reserpine
Acronycine
OCH,
Q CH,
Caffeine
Sanguinarine
OCH,
CH.O
^N
Quinine
CH2
H?C-CCHCH = CH2
2 H
-------
21'
47 20'/CH3
Vinblastine R=-CH3 Vincristine
Table LI (p.2 of 2)
-------
Table LII
Physicochemical Properties of Some Plant Alkaloids'
Compound
m.p. (°C) [0(]D (solvent)
Solubility
Resetpine
264-265
Sanguinarine
Nicotine'(liquid)
Acronycine
Emetine
190-191
Caffeine
Quinine
Vinblastine
Vincristine
238
177
-118° (chloroform) Insoluble in water; soluble
in chloroform, dichloro-
methane, acetic acid, ben-
zene and ethyl acetate;
slightly soluble in methanol
and ethanol
Soluble in ethanol, chloro-
form, acetone and ethyl
acetate
-169C
-50° (chloroform)
-169° (ethanol)
284-285 -28° (methanol)
273-281 +8.5° (methanol)
Soluble in ethanol, chloro-
form and ether
Sparingly soluble in water;
soluble in organic solvents
Sparingly soluble in water;
soluble in chloroform,
methanol, ethanol, ether and
acetone
Soluble in water, chloro-
form, ethanol, acetone and
benzene
Slightly soluble in water;
soluble in ethanol, chloro-
form, benzene and glycerol
Soluble in water and chloro-
form; slightly soluble in
ethane; insoluble in diethyl
ether
Soluble in water and chloro-
form; slightly soluble in
ethanol; insoluble in di-
ethyl ether
Summarized from IARC Monographs, Vols. 24 and 26, International Agency for
Research on Cancer, Lyon, France, 1980 and 1981; The Merck Index, 10th ed.,
Merck and Co., Rahway, N.J., 1983.
bSee Table LI for structural formulas.
-------
macological properties of reserpine (109), quinine (110), nicotine (112) and
caffeine (1, 113) have been reviewed. It has become clear that the antihyper-
tensive and sedative effects of reserpine are due to depletion of stores of
catecholamines and 5-hydroxytyrosine in the brain and adrenal medulla. The
primary antimalarial action of quinine is schizontocidal. In addition, thera-
peutic doses of quinine cause analgesia and antipyresis and have curare-like
effects on skeletal muscle. Nicotine exerts actions on the central and peri-
pheral nervous systems, the cardiovascular system, the gastrointestinal tract
and on exocrine glands. In general, the effects of this alkaloid have both
stimulant and depressant phases. Five principal pharmacological actions of
caffeine are known: respiratory stimulation, skeletal muscle stimulation,
diuresis, cardiac stimulation, smooth muscle relaxation, and central nervous
system effects.
Toxic effects. Many of these alkaloids are highly toxic to rodents.
Their LDcn values in the rat, mouse or rabbit are shown in Table LIII.
Undesirable effects of reserpine observed in patients are primarily asso-
ciated with the gastrointestinal tract and the central nervous system. The
following untoward responses are common: abdominal cramps, diarrhea, ulcer,
insomnia, nightmares and depression. Single parenteral doses of reserpine
produce gastric haemorrhage and erosion (122) and suppress the immune response
of lymph node cells (123) in mice.
Sanguinarine is the active poison in argemone seed oil. Epidemics of
poisoning from argemone seed oil have been frequently recorded in India. The
most common effects include dropsy, diarrhea, edema of the legs, glaucoma,
anaemia, fever, redness of the skin, alopecia and dyspnea. All these toxic
effects have been experimentally produced in animals with sanguinarine
296
-------
Table LIII
Acute Toxicity of Some Plant Alkaloids8
Compound
Reserpine
Sanguinarine
Nicotine
Emetine
Caffeine
Quinine
Vinblast ine
Vincristine
Species and route
Rat , i .v .
Mouse, oral
i.p.
Mouse, i.p.
Rat , oral
i .v .
Rat , oral
Rat , oral
Rabbit , oral
Rat , i.p.
i.v.
Mouse, oral
i.p.
i .v .
Rat , i.p.
i .v .
Mouse, i.p.
i .v .
LD^Q (mg/kg)
18
500
70
18
55
1.0
12.1
200
800
2.2
2.9
15
5.6
10
1.2
1.0
4.7
1.7
Reference
(108)
(115)
(115)
(116)
(117)
(117)
(118)
(117)
(117)
(119)
(120)
(119)
(119)
(120, 121)
(119)
(120)
(119)
(121)
aSee Table LI for structural formulas,
-------
(116). In vitro studies with the pigeon brain homogenates showed that san-
guinarine hydrochloride inhibits some -SH group-containing enzymes (124).
Both acronycine and emetine have cytotoxic and antineoplastic proper-
ties. Acronycine inhibits the synthesis of nucleic acids by interferring with
the transport of nucleosides across the cell membrane (125, 126). Subchronic
i.p. administration of acronycine to rats and mice at doses higher than 25
mg/kg resulted in high mortality. At lower doses, inflammation and fibrosis
of the peritoneal cavity were noted (127). Bnetine is a potent protein
synthesis inhibitor in eukaryotes by blocking the transfer of amino acids from
t-RNA to the polypeptide chain being formed (128). Structure-activity rela-
i %
tionship studies on a number of benzoisoquinoline alkaloids indicate that a
planar molecule with 2 aromatic rings and the presence of a nucleophilic
element, such as a nitrogen atom at a certain distance from the aromatic
rings, are required for protein synthesis inhibitory activity. The distance
between the 2 aromatic rings, the angle between the nitrogen atom and the
rings, the electronegative character of the rings and the planarity of the
structure are important features in determining the activity (129). Clinical
toxic manifestations of emetine include nausea, diarrhea, vomiting, epidermal
inflammation, aching, tenderness and weakness of muscle and effects on the
cardiovascular system (110).
The toxic response of vinblastine and vinicristine are quite different
despite their similarity in chemical structures. Vincristine is more toxic in
animals and produces, more frequently, peripheral neuropathy, abdominal pain,
alopecia and liver impairment. The most important toxic effect of vinblastine
is leukopenia. The cytotoxic activity of these agents are related to their
ability to inhibit the formation of microtubules of the mitotic spindle,
resulting in arrest of dividing cells in metaphase. Structure-activity rela-
297
-------
tionship analysis reveal that either hydrogenation of the double bonds, reduc-
tive formation of carbinols, removal of the acetyl group at C-4 or acetylation
of the hydroxyl group diminishes the cytotoxicity of vinblastine and vincris-
tine (111). The configurations at C2" and CIS1 as well as the presence of the
methoxycarbonyl group on CIS1 (see Table LI) also play an important role in
determining biological activity (130).
Quinine is a strong local irritant. The acute toxic effects of quinine
in humans are characterized by a spectrum of symptoms referred to as
"cinchonism." These symptoms involve hearing and vision, the gastrointestinal
tract, the nervous, cardiovascular and renal excretory systems, and the
skin. The fatal oral dose of quinine for humans is about 8 grams (110).
Nicotine can be rapidly absorbed through skin and mucous membranes. The
major symptoms of acute nicotine poisoning in humans include nausea, vomiting,
abdominal pain, diarrhea, cold sweat, headache, mental confusion and convul-
sions. An oral dose of 40 rag is fatal for an adult. Death may result from
respiratory failure caused by paralysis of muscles of the respiratory system.
In humans, overdoses of caffeine (15 mg/kg or more) cause pharmacological
responses predominantly in the central nervous system and the circulatory
system insomnia, restlessness, excitement, sensory disturbances, tachycar-
dia and extrasystoles. Lower doses may cause nausea, nervousness, insomnia
and diuresis (113, 117). Caffeine is cytotoxic and affects the mitotic rate
of a wide range of cell types (rev. in 107).
Mutagenic effects. The genotoxic potential of these plant alkaloids has
been investigated in a number of systems. The results, which are summarized
in Table LIV, indicate that many of them possess clastogenic properties but
are probably not potent mutagens.
298
-------
Table LIV
Mutagenic and Related Genotoxic Activities of Some Plant Alkaloids3
Compound
Reserpine
Chromosomal
Ames test aberrations
- (131-133) + (143)
- (144)
Other test0
- [A-D] (132, 133, 154, 155)
Sanguinarine + (134)
Vinblastine - (135, 136)
Vincristine - (135)
Quinine
Nicot ine
Caffeine
+ (137)
- (138)
- (139-141)
- (136, 137,
139, 142)
+ (145) + [E.F] (136, 146)
- (146) - [D.G-J] (155-158)
+ (147) + [K.K.L] (149, 159, 160)
- (148-150) - [I.J.L] (149, 157, 158, 161)
+ (138) - [F.M.N] (137)
+ [F.L] (138)
+ [F.L.M] (141, 162)
+ (151)
- (151)
+ (152, 153)
+ [A.K-M] (107, 151, 163, 164)
- [D,F,J,K,M,N] (137, 155, 165-
169)
"»" * positive; "-" = negative; numbers in parenthesis are references.
See Table LI for structural formulas.
A " Bacillus subtilus; B s Aspergillus nidulans; C = Unscheduled DNA syn-
thesis; D » Dominant lethal assay; E » Sperm abnormality test; F = Micro-
nucleus formation; G * Chlamydomonas reinhardi; H = Schizosaccharomyces pombe;
I « host-mediated assay; J » Chinese hamster cell/HGPRT assay; K = Mouse
lymphoma cell/L5178Y; L * Sister-chromatid exchange; M » Escherichia coli;
N « Sex-linked recessive lethal in Drosophila.
Pyrolysate.
Hydrochloride or dihydrochloride salt.
-------
In the Ames Salmonella assay, only the pyrolysate of sanguinarine (134)
and the dihydrochloride salt of quinine (137) gave positive results when
tested in the presence of S-9 fraction; other alkaloids were negative either
with or without metabolic activation (131-133, 135-137, 139-142, 149).
Reserpine was also non-mutagenic in Bacillus subtil is (132) and in
Aspergillus nidulans (154). It did not induce dominant lethality in mice
(155) or unscheduled DNA synthesis in primary rat hepatocytes (133). Cyto-
genetic studies on human lymphocytes in culture (132, 151), in Chinese hamster
cells in vitro, or in bone marrow cells from rats treated in vivo (132) did
not reveal any significant clastogenic activity of reserpine. However,
f ^
Jameela and Subramanyam (143) have reported that reserpine causes chromosomal
aberrations in meiotic cells of grasshoppers and in bone marrow cells of mice.
The genotoxic effects of vinblastine and vincristine have been reviewed
(170-172). Most studies showed no mutagenic and related genotoxic activity of
these vinca alkaloids. Negative responses were observed for vinblastine in
the forward mutation assay in Schizosaccharomyces ppmbe and in the backward
mutation assay in Chamydomonas reinhardi (156). Neither vinblastine nor
vincristine displayed mutagenic action in host-mediated assays in rats (149,
157), in dominant lethal assays in mice (155, 156) or in V79 Chinese hamster
cells in vitro (158). No increase in chromosomal aberrations (146, 148-150)
or in sister chromatid exchange (161) were found by some authors with vincris-
tine in several test systems. Other investigators, however, have reported
that vincristine is mutagenic in mouse lymphoma L5178Y cells (159) and induces
micronucleus formation (146, 149), sister chromatid exchange (160), chromo-
somal translocations (173) and other chromosomal aberrations (147). Vinblas-
tine produces increase in chromosomal translocations (173), bone marrow micro-
nucleus formation and sperm abnormalities in mice (136), in addition to
299
-------
various chromosomal aberrations in cultures of a cell line from the lung of
the Chinese hamster (145).
Quinine dihydrochloride is a frameshift mutagen in strains TA98 and
TA1538 of Salmonella typhimurium in the presence of S-9 mix, but shows no
responses in the following test systems: Escherichia coli, host-mediated
assay in mice, sex-linked recessive lethal test in Drosophila melanogaster and
micronucleus test in bone marrow cells of mice (137). Quinine hydrochloride,
on the other hand, appeared inactive in the Ames test but showed a dose-
dependent increase of sister chromatid exchange, enhanced incidence of micro-
nuclei, and elevated chromatid breaks (138).
No mutagenic effect was observed with nicotine in the Ames test (139-
141). Cytogenetics studies on human leukocytes in vitro did not disclose any
clastogenic activity of nicotine (151). However, the alkaloid induces DNA
damage in the E. coli pol A+/A~ system (141), chromosomal aberrations in mice
in vivo (151) and sister chromatic exchange in Chinese hamster ovary cells
(162).
No information on the mutagenic activity of acronycine is available.
finetine was reported to exhibit a mutagenic effect when tested in
Corynebacterium (cited in 174).
Concern over the genetic hazards involved in the consumption of caffeine-
containing beverages have stimulated much research on the mutagenic and
related genotoxic activities of caffeine in recent years. The possibility
that caffeine might be an environmental mutagen is a subject of many reviews
(e.g., 107, 175, 176). Although there exists a series of negative results in
a variety of assay systems (e.g., 136, 137, 139, 142, 155, 165-169), the
mutagenic and clastogenic actions of caffeine in systems ranging from micro-
300
-------
organisms and plant cells, to mammalian cells had been established more than
twenty years ago (see 107, 175, 176). Recent research has also shown that
caffeine induces high frequency of mutations in Bacillus subtilis (163), point
mutations and chromosomal breakage in mouse lymphoma cells (153), sister
chromatid exchanges in mice in vivo (164), and various types of chromosomal
aberrations in somatic ganglia of Drosophila melanogaster (152). Furthermore,
a number of studies demonstrate that caffeine has a synergistic effect on the
mutagenic and chromosome-damaging activities produced by other chemical
mutagens or by radiation (e.g., 177-182). Since caffeine inhibits the activ-
ity of DNA polymerase I (183) , it is believed that inhibition of excision
repair of DNA by caffeine i'S ^responsible for its potentiation of mutagenicity.
Structure-activity relationship analysis indicate that caffeine
(1,3,7-trimethylxanthine) is more clastogenic in human lymphocytes in culture
than 1,3-dimethyl- or 3,7-dimethyl-xanthine; 1,7-dimethyl, 1-methyl-,
3-methyl-, and 7-methyl-xanthines are not clastogenic (184). Caffeine is also
more active than its 8-substituted analogs (8-methoxy-, 8-ethoxy-, or
8-chloro-caffeine) as a mutagen in Jj_. coli or as a co-clastogen with thio-TEPA
(triethylenethiophosphoramide) or maleic hydrazide in the production of
chromosomal aberrations in Chinese hamster cells and Vicia faba root tips
(107).
Teratogenic effects. Reserpine is teratogenic in the rat. Spina bifida
and eye defects were induced in the offspring of rats administered 0.8-1.5
mg/kg body weight reserpine on day 9, or 1.5-2.0 mg/kg body weight on day 10
of gestation (185). When pregnant rats were parenterally administered 1 mg/kg
body weight reserpine daily for 3 days during the last week of gestation,
hydronephrosis and deformities of the brain ventricles developed in the
newborns (186). In addition, reserpine affects the reproduction and the
301
-------
A A,
postnatal neuroendocrine function of rodents (rev. 108). In humans, a
A
correlation with malformation has been implicated in a study of 475 pregnant
women treated with reserpine and other anti-hypertensive drugs (see ref.
108). There are also reports of nasal congestion with cyanosis, costal
retraction, lethargy, congenital lung cysts and stillbirth of babies whose
mothers were treated with reserpine during pregnancy (187, 188).
The teratogenic action of antimitotic agents is well documented (see
171, 189). Vinblastine has been shown to be teratogenic in the mouse (190),
the rat (191, 192) and the hamster (193). Evidence for the teratogenicity of
vincristine has been found also in the monkey (194), the mouse (147, 195), the
t ^
rat (196) and the hamster (193). In general, both alkaloids induce a similar
pattern of effects which include fetal mortality, growth retardation, skeletal
defects and malformations of a wide variety of organs. The most effective
teratogenic doses of vinblastine and vincristine in these animals are between
0.1 mg/kg and 0.25 mg/kg body weight. In several case reports, no birth
defects were found in infants of women receiving various doses of these
alkaloids during pregnancy (rev. in 171).
Growth retardation, delay in teeth eruption and in eye opening, and
various congenital malformations were noted in the progeny of female rats
ingesting quinine (0.25 mg/ml) from drinking water during the pre-gestative,
gestative and lactating periods (197). Robinson _et__al_. (198) reported that
two of 200 women treated with quinine during early pregnancy gave birth to
congenitally deaf babies. In 21 cases of attempted abortion by taking large
doses of quinine, congenital malformation involving the central nervous
systems, limbs, face and the digestive and urogenital systems resulted
(199). Abortion may also result from sanguinarine poisoning (116).
302
-------
Nicotine, at doses of 0.4, 1.5 and 5.0 mg/kg body weight, has adverse
effects on the formation of the cardiovascular system and ossification of the
skeleton in embryos of Wistar rats (200). Nishimura and Nakai (201) found
skeletal defects and cleft palates in the newborns of mice administered
nicotine at 25 mg/kg body weight on days 9, 10 and 11 of gestation.
There was no significant teratogenic response in rats injected i.p. with
emetine at 5.0 mg/kg body weight on the 12th day of gestation. However, this
inhibitor of protein synthesis potentiates embryolethality and teratogenicity
of caffeine and other teratogens (202, 203).
Much information on the Xeratogenicity of caffeine was obtained from
experimental studies in rats, mice, rabbits and hamsters. Thayer and Palm
(176) reviewed extensively the teratogenic potential of caffeine and have
tabulated the findings reported between 1960 and 1974. The subject has also
been covered in update reviews in 1977 (107) and in 1981 (114). Several
recent studies confirm most of the earlier observation that at high doses,
caffeine is teratogenic in animals. In Charles River CD1 mice, a single dose
of 100 mg/kg caffeine injected intraperitoneally on day 14 of pregnancy or
single oral doses of caffeine of 200 and 300 mg/kg caused cleft palate in some
fetuses (204). Low incidence of retarded skeletal ossification, missing or
hypoplastic nails and cleft palate was observed in fetuses of pregnant mice
and rats given about 150 mg/kg caffeine in the drinking water (205, 206).
Young and Kimmel (207) also noted a dose-related increase in skeletal malfor-
mations in the offspring of rats treated intravenously with caffeine on day 11
of gestation at 112.5 mg/kg or 150 mg/kg. Moreover, the potentiating effect
of caffeine on the teratogenicity of other agents have been reported (e.g.,
202, 208, 209).
303
-------
Caffeine and its metabolites readily cross the human placenta (210). An
increase in the half-life of the excretion of caffeine has been reported in
women during pregnancy (211). However, a number of studies which attempted to
correlate the intake of caffeine with birth defects in humans showed no
definite causal relationship (rev. 114). Nonetheless, an inordinately high
incidence of abortion or stillbirth was noted in a subgroup of 16 women during
a retrospective survey involving 800 women, three-fourths of whom were
Mormons. The 16 women in the subgroup were identified as having an estimated
daily intake of caffeine of 600 mg or more (212). A recent nationwide case-
control study in Finland, comparing the mothers drinking at least four cups of
coffee a day during pregnancy, with those not drinking coffee at all, showed a
relative risk of coffee consumption with respect to congential malformation
(213).
Several short-term teratogenesis assays have also disclosed the terato-
genic action of reserpine, quinine, vinblastine, nicotine and caffeine (214-
216).
5.3.2.3.2.3 CARCINOGENICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
5.3.2.3.2.3.1 Carcinqgenicity of Pj.ant Alkaloids Other Than Pyrrolizidine.
The carcinogenesis studies of some plant alkaloids are summarized in Table
LV. Positive carcinogenic effects in experimental animals have been reported
with reserpine, sanguinarine, nicotine, acronycine, and most recently with
caffeine. In limited studies, no evidence of carcinogenicity was found in
rats or mice following chronic exposure to emetine, quinine, vinblastine, or
vincristine.
Reserpine. Reserpine is carcinogenic in rats and mice. In a two-year
bioassay, in which groups of 50 F344 rats and 50 B6C3Fj mice of each sex were
304
-------
p. 1 of 2
Table LV
Carcinogenicity of Some Plant Alkaloids
Compound3
Reserpine
Sanguinarine
(or argemone
oil)
Nicotine
Acronycine
Emetine
Caffeine
Species and strain
Rat, Fischer 344
Rat , Wistar
Rat, Wistar
Mouse, BSCSFj
Mouse, C3H; XVIInc
Rat ,
! ^
Rat, mouse, hamster
and guinea pig
Mouse, Swiss
Rat, Wistar
Mouse, Swiss
Rat , Sprague-Dawley
Mouse, B6C3?!
Rat , Sprague-Dawley
Mouse, B6C3Fj
Rat , Sprague-Dawley
Rat , Wistar
Rat , Wistar
Mouse, C57BL/6
Route
oral
oral
oral
oral
oral
implantation
i.v .
topical
oral
oral
i .p.
i.p.
i .p.
i.p.
oral
oral
oral
oral
Principal organs
affected
Adrenal gland
Liver, hematopoietic
t issue
Noneb
Seminal vesicle,
mammary gland
None0
Bladder
(Sarcomas)
Skin
Intestine, liver
None
Mammary gland, bone
and peritoniura
None6
Nonef
Nonee'f
None
None
Pituitary gland
None
Reference
(115)
(217)
(218)
(115)
(219)
(116,
220, 221)
(116)
(116,
220, 221)
(222)
(223)
(127)
(127)
(224)
(224)
(225,
226)
(227)
(228)
(229)
-------
Table LV (continued)
p. 2 of 2
Compound3
Quinine
Vinblastine
Vincristine
Species and strain
Rat, Leeds
Mouse, Stock
Rat , BR-46
Rat , Sprague-Dawley
Mouse, Swiss
Rat , Sprague-Dawley
Mouse, Swiss
Route
oral
intravaginal ,
bladder
implantation
i.v .
i.p.
i.p.
i.p.
i.p.
Principal organs
affected
None
None
None
None
None
None
None
Reference
(230)
(231,
232)
(233,
234)
(235)
(235)
(235)
(235)
aSee Table LI for structural formulas.
Treatment for only 75 weeks.
cAt a dose level of 0.24 jig/day.
^Nicotine pyrolysates or nicotine hydrochloride.
eHigh early mortality rate of treated animals.
^Experiment terminated after 84 weeks.
-------
administered reserpine in the feed at doses of 5 ppm or 10 ppm, dose-related
neoplasms occurred in both species. Adrenal medullary pheochromocytomas were
induced in 24 of the 48 surviving the high-dose, and in 18 of the 49 low-dose
male rats. In the mice, 7 cases of mammary carcinomas were found in 48 high-
dose as well as in 49 low-dose females; carcinomas of the seminal vesicles,
which were not seen in 50 control males, developed in 5 of the 49 high-dose
and in one of the 50 low-dose males (115). Low incidences (13-16%) of
hepatomas and lymphosarcomas were reported in groups of 43-50 male and 80-92
female Wistar rats receiving reserpine (100 mg/kg) in a semi-liquid diet for
18 months (217). However, administration of 30 or 60 mg/kg body weight of
dietary reserpine to groups -of 25 male and 25 female Wistar rats for 75 weeks
did not result in significant tumor incidence (218). Lacassagne and Duplan
(219) detected no tumorigenic activity of reserpine in a group of 24 female
C3H mice and in a group of 11 female XVIInc mice, receiving an average of 0.24
iig reserpine per day in the diet for life. The failure of the last two
experiments to confirm the carcinogenic activity of reserpine may have been
due to the short exposure period and the low dose used.
Sanguinarine. Hakim (116, 220, 221) has established in several experi-
ments that sanguinarine and its major metabolite, benz[c]acridine, are
complete carcinogens, inducing bladder tumors in rats and skin tumors in
mice. The author induced tumors of the bladder by implanting paraffin pellets
(15 mg) containing 25% sanguinarine or benz[c]acridine into the bladder of
rats. Skin tumors were induced by repeated painting with solutions of san-
guinarine or benz[c]acridine on the skin of mice. Metastasizing sarcomas have
also been produced in rats, mice, hamsters and guinea pigs with a single i.v.
injection of 0.05 to 0.1 ml argemone oil (containing about 0.1 mg sanguin-
arine). The carcinogenicity and structure-activity relationships of benzacri-
dines have been discussed in Section 5.1.1.4, Volume IIA.
305
-------
Nicotine. Truhaut and DeClercq (222) have described the development of
malignant teratomas and chronic inflammatory lesions in the intestine and
liver of some Wistar rats 12-17 months following ingestion of nicotine or
nicotine pyrolysates in the drinking water at a dose level of 10 mg/kg body
weight. Control animals or rats s.c. injected with 5 mg/kg nicotine
pyrolysate once weekly for life did not bear such tumors. The authors and
associates (236, 237) further showed that cotinine (see Fig. 12 for structural
formula^, the major metabolite of nicotine, is also carcinogenic inducing
lymphoid sarcomas or lymphoid leukemia in the alimentary tract, the liver, the
lung and the spleen in 12 of 15 rats given cotinine (500 rag/liter) in the
drinking water for 8-18 months. Schmahl and Osswald (238), however, failed to
confirm the carcinogenic action of cotinine in a group of 100-day-old Wistar
rats given approximately 30 mg/kg body weight of cotinine in the drinking
water for 17-21 months. Administration of nicotine hydrochloride to groups of
50 male and 50 female Swiss mice (5-6 weeks old) in the drinking water at a
concentration of 0.0625% or 0.09375% for life, did not produce a significant
tumor incidence (223). There was also no tumorigenic effect of nicotinic acid
(pyridine-fi-carboxylic acid), a vitamin that occurs in legumes, corn and
other plants, under similar study conditions (223).
Nicotine sulfate, which was used together with copper sulfate as a drench
to combat parasites on a large farm in South Africa from 1952 to 1962, was
incriminated in the high incidence of esophageal tumors in sheep (239).
Acronycine. Acronycine is carcinogenic in Sprague-Dawley rats, inducing
tumors of the mammary gland in females, osteosarcomas in males, and sarcomas
and other tumors of the peritoneum in both sexes. These findings arose in a
chronic study in which groups of 35 rats of each sex were administered acro-
nycine 3 times weekly by i.p. injection at a dose of 3.75 mg/kg body weight
306
-------
for about one year. In groups of B6C3Fj mice, receiving either 2, 6, 12.5 or
25 mg/kg acronycine, the high mortality rates precluded an evaluation of the
carcinogenic effect of acronycine in this species (127). The mouse pulmonary
tumor assay, in which a total dose of 0.5, 1.3 or 2.6 g/kg acronycine was
given to A/He mice in 5 i.p. injections over 8 weeks, did not reveal the car-
cinogenic effect of acronycine (240).
Emetine. The carcinogenic potential of emetine has been studied in
Sprague-Dawley rats and B6C3Fi mice by administering the alkaloid via i.p.
injection at doses of 0.5 or 1 mg/kg body weight for rats and 1.6, 3.2 or 6.4
mg/kg body weight for mice 3 times/week for up to 52 weeks. At the termina-
i- ^
tion of the studies (at week 83 or 84), no tumors occurred at a statistically
significant incidence in treated rats'or mice compared with controls. How-
ever, it was noted that the survival of the treated mice was low in this
study. The study was conducted only for 84 weeks instead of two years (224).
Emetine did not exhibit a positive response in the mouse pulmonary tumor
assay at the dose levels of 40, 100 and 185 mg/kg body weight (240).
Caffeine. Caffeine was found to be non-tumorigenic in animals by several
investigators. No significant increase in tumor incidence was found in mice
or rats given caffeine in the diet (225, 229) or in the drinking water (226,
227) for up to two years, at doses exceeding the maximum tolerated level.
Similarly, oral administration of freshly brewed or instant coffee to mice or
rats for life did not result in higher incidence of tumors in various organs
(241-244). However, Yamagami et_ al_. (228) reported in 1983 that caffeine
caused pituitary tumors in female Wistar rats. Microadenomas, papillary (or
sinusoidal) macroadenomas, and diffuse macroadenomas or hyperplasia of the
pituitary were found in 27 of the 40 rats receiving caffeine at a concentra-
307
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tion of 2 mg/ml in the drinking water for 12 months. Such lesions were found
only in 9 of 30 control rats.
Quinine. There is no evidence that quinine is carcinogenic. Early
experiments conducted by Boyland and associates (231, 232) did not yield
significant incidence of neoplasms of the uterine cervix or the bladder in
mice following application of quinine sulfate intravaginally or by implanta-
tion of the compound in the urinary bladder. More recently, the effects of
chronic oral dosing with quinine sulfate in the rat have been examined by
Flaks (230). A group of 48 male albino rats of Leeds strain received 0.1%
quinine sulfate in the drinking water for up to 15 months. No tumors or pre-
t ^
neoplastic lesions were noted in the rats autopsied during the course of the
experiment or in the 13 survivors after 15 months of treatment.
Vinblast ine. Vinblastine did not produce significant tumor incidence in
rats or mice wnen tested by i.v. or i.p. administration. In a group of 36
male BR46 rats given i.v. injections of 0.33 mg/kg body weight (17% of the
LDCQ) vinblastine sulfate once every two weeks for 10 weeks, a 12% tumor inci-
dence was found. However, 11% had tumors among the 689 controls (233, 234).
In another study, in which 48 male BR46 rats were injected intravenously with
0.14 mg/kg body weight vinblastine sulfate once weekly for 52 weeks, only one
of the 25 surviving rats bore a benign thymoma 18 months after start of
treatment (233). When groups of 25 Sprague-Dawley (CD) rats and 25 Swiss-
Webster mice of either sex were administered the compound by i.p. injection, 3
times weekly for 26 weeks at doses of 0.1 and 0.2 mg/kg body weight (rats) or
0.09 and 0.18 mg/kg body weight (mice), the tumor frequencies in the treated
animals were not statistically different from the control values (52% vs. 34%
in male rats; 72% vs. 58% in female rats; 16% vs. 26% in male and female mice)
(235).
308
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Vincristine. As with vinblastine, there is no evidence for carcinogenic
activity in rats and mice after i.p. administration of vincristine. The tumor
incidences in groups of 25 Sprague-Dawley (CD) rats and 25 Swiss-Webster mice
of each sex, given i.p. injections of vincristine sulfate at 0.06 and 0.12
mg/kg body weight (to rats) or 0.075 and 0.15 mg/kg body weight (to mice) 3
times/week for 26 weeks, were not significantly higher than those in the
controls (235). The compound did not produce morphological transformation of
mouse C3H/10Tj/2 clone 8 cells (148) or hamster embryo cells (245) .
5.3.2.3.2.3.2 Modification of Carcinogenesis. Experimental studies in
animals have demonstrated both enhancing and protecting effects of plant
i ^
alkaloids upon carcinogenesis with other chemicals, depending on the time of
treatment and dose level.
Reserpine, for instance, stimulated the induction of mammary tumors by
7,12-dimethylbenz[a] anthracene (CUBA) or by N-nitrosomethylurea (NytU) in the
rat, when it was given after the administration of the carcinogen; however,
when reserpine was administered before or concurrently with EMBA or N4U, it
suppressed mammary tumorigenesis (246, 247). Retardation by reserpine of
hepatocarcinogenesis by diethylnitrosamine in rats (248) and of mouse skin
tumorigenesis by 3-methylcholanthrene (249) was observed when the animals were
treated with reserpine and the carcinogen simultaneously.
Nicotine has long been suspected to be one of the cocarcinogens in
cigarette smokers. In studies using the two-stage mouse skin model, nicotine
promotes the tumorigenesis of EMBA. It enhances, at dose levels between 2.5
and 5.0 mg/kg body weight, and inhibits, at higher doses, the carcinogenic
activity of mixtures of benzopyrene and 12-0-tetradecanoylphorbol-13-acetate
(TPA) on the mouse skin (250). A tumorigenesis-promoting activity has also
309
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been observed with cotinine but not with nicotine-1'-N-oxide. It was sug-
gested that metabolism of nicotine to cotinine might account for the cocar-
cinogenic effect and metabolism to nicotine-1'-N-oxide might account for the
inhibitory effects of nicotine at high doses (250). Nicotine has also been
shown to enhance stomach carcinogenesis by N-methyl-N'-nitro-N-nitrosoguani-
dine (MNNG) in the rat; combined treatment of rats with MNNG and nicotine led
to an earlier development and an increased incidence of stomach tumors (251).
Welsch j^t__al_. (252) observed that the administration of caffeine in the
drinking water (250 and 500 mg/1) to female Sprague-Dawley rats, 3 days after
treatment with EMBA (5 mg, i.g.) for 21 weeks, resulted in an increase in
mammary carcinoma incidence. The same effect was found when caffeine was
given to the rats for 6 weeks beginning 20 weeks after exposure to EMBA.
However, administration of caffeine prior to and during EMBA treatment did not
significantly affect mammary tumor incidence in the rats. On the other hand,
caffeine markedly increased the incidence of papillomas on the skin of mice,
when it was given 6-9 hours before urethan administration. Application of
caffeine to the skin of mice 6 hours after urethan, however, led to lower
incidence of skin tumors (253). Nomura (254) found that when lung tumors were
induced in young adult mice or in mouse fetuses by s.c. injection of either
urethan or 4-nitroquinoline-l-oxide, the tumor incidences were significantly
reduced by caffeine after treatment with the carcinogen. The caffeine-
sensitive period for suppression of lung neoplasia was found to parallel the
generation time of the stem cells in the lung of mouse fetuses and young adult
mice. It has been suggested that the inhibition by caffeine of the error-
prone post-replication repair of DNA or the alteration of the metabolism of
carcinogens by caffeine may be the mechanism of the generally observed modifi-
cation of chemically-induced neoplasia by caffeine (253, 254). The effects of
310
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caffeine on the hepatic mixed-function oxidases in various species have been
described (255) .
Ranadive et al. (256) observed, in the mouse, the cocarcinogenic effect
of argemone-oil (which contains sanguinarine as the principal constituent) and
of a market sample of mustard oil from India.
Recent research indicates that many chemicals, which induce viral gene
expression in human lymphoblastoid cells latently infected with Epstein-Barr
virus (EBV), are mouse skin tumorigenesis promoters. Although vinblastine
possesses EBV-activating property, it fails to show any significant tumori-
genesis- promo ting activity i,n^the mouse skin (257).
5.3.2.3.2.4 METABOLISM AND POSSIBLE MECHANISMS OF ACTION
The pharmacokinetics and metabolic fate of many of these alkaloids have
been investigated in various laboratory animals as well as in humans. Except
for sanguinarine and nicotine, metabolic activation does not appear to be
required for their biological and carcinogenic activities.
Reserpine. Reserpine is rapidly absorbed and metabolized in most tissues
of rats, mice, dogs and rhesus monkeys. In the rat, reserpine is hydrolyzed
to methyl reserpate which is excreted primarily in the urine (258). In the
mouse, the major urinary metabolites after oral or i.v. administration of
reserpine is trimethoxybenzoic acid (259).
As reserpine has not been shown to be mutagenic, a possible mechanism by
which reserpine exerts its mammary carcinogenic effects may be via certain
endocrine functions. In rodents, reserpine administration has been
demonstrated to be associated with elevated levels of serum prolactin and it
is known that a correlation exists between the duration and extent of increase
in prolactin levels and the development of mammary neoplasms (252, 260).
311
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Recently, it has been shown that chronic reserpine treatment increases the
mammary tumor estrogen receptor and peroxidase activity in the rat, indicating
that there is increased estrogenic stimulation of specific protein production
(247). Furthermore, reserpine has been shown to impair the immune system of
rats, inhibit oxidative phosphorylation in isolated mitochondria, and to
interfere with transport systems in the cell membrane (see ref. 261).
It is interesting to note that unlike reserpine, yohimbine, another
indole alkaloid, which differs from reserpine in lacking an acetyl group and
the trimethoxybenzoic moiety in the molecule, does not modify chemically
induced carcinogenesis in the liver of the rat (262).
Sanguinarine. In the rat, sanguinarine is readily absorbed after feed-
ing, is stored in the liver, and is biotransformed to at least four different
fluorescent metabolites which are excreted as protein complexes in milk, bile
and urine. Following parenteral administration into five species of animals,
this carcinogenic alkaloid distributes into the stomach and esophagus where
metabolism occurs. One of the metabolites has been identified as benz[c]acri-
dine (116). The carcinogenic activity of sanguinarine and benz[c]acridine may
be related to their intercalation into DNA (263, 264; see also Notes to
Section 5.1.1.6.2.3, Volume HA).
Nicotine. Nicotine is well absorbed through the respiratory tract,
intestine and skin. Radioactivity was detectable in the bronchial wall, the
melanin-containing tissues and in the urinary bladder wall, for up to a month
after i.v. injection of 1^C-methyl- or 2'-^C-labelled nicotine into the mouse
(265). The compound is metabolized mainly in the liver but also in the lung
and kidney. The major metabolites are cotinine, formed by oxidation at the
o^-carbon, and nicotine-1'-N-oxide, formed by N-oxidation of the pyrrolidine
312
-------
ring (266). N-Demethylated metabolites and CC^ are produced in the lung (267-
269). Nicotine and its metabolites are mainly excreted through the kidney;
they have also been detected in the milk of lactating women who smoke (270).
Since both carcinogenic and cocarcinogenic activity have been shown for
cotinine (see previous Sections), the biotransfonnation of nicotine to
cotinine appears to be important for the carcinogenic action of nicotine. It
has been postulated (271) that oxidative N-demethylation of nicotine (or coti-
nine) may yield chemically active N-methyleniminium species that can interact
with cellular nucleophiles (see Fig. 12).
Acronycine. Metabolic studies of acronycine in rats, mice, dogs, cats,
r %
rabbits, guinea pigs and humans have shown that hydroxylation of the compound
at C-9 and C-ll (see Table LI) occurs in all species. The gem-dimethyl groups
(C-3) of acronycine are hydroxylated in rats, mice, dogs and humans but not in
guinea pigs. The mouse and the guinea pigs also metabolize acronycine by
0-demethylation (272).
The mechanism(s) of the carcinogenic action of acronycine is obscure. It
is not known if its interaction with cell-surface components and interference
with some transport systems on the cell membrane (125, 126) may result in
impaired cellular activities leading to alterations of gene expression. On
the other hand, acronycine and its metabolites may act by intercalation with
DNA by virtue of the acridine moiety in the molecule.
Emetine. Following parenteral administration, the alkaloid can be found
in the liver and, to lesser extent, the lung, kidneys and spleen of humans.
Emetine is metabolized slowly; considerable levels of the compound can still
be found in the urine 40 to 60 days after treatment (110).
313
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^N^ CHU
[O] ^
CH2OH
[o]
o
CH2OH
O
Fig. 12. Proposed metabolic pathway for the activation of nicotine.
-------
Caffeine. The pharmacokineti.es and metabolism of caffeine in humans and
animal species have been reviewed (107, 114). Recent investigations (273,
274) confirm that the major pathways in the metabolism of caffeine in humans
are N-demethylation and ring oxidation to paraxanthine (1,7-dimethylxanthine),
theophylline, theobromine and 1,3,7-trimethyluric acid. These compounds are
further degraded to dimethylated uric acids, monomethylated uric acids, and
monomethylxanthines. An acetylated metabolite, 5-acetylamino-6-amino-3-
methyluracil, has also been detected in the urine of humans after oral uptake
of caffeine (274, 275). In the mouse, the major urinary metabolites of
caffeine arc 1,3,7-trimethyluric acid, 1,3-dimethyluric acid, 1-methyluric
acid, 1,7-dimethylxanthine, 3-methylxanthine and 6-amino-5-(N-formylmethyl-
amino)-!,3-dimethyluracil (114, 276). Metabolism of caffeine in rats also
involves hydrolyt'ic ring-opening, resulting in the formation of diaminouracil
derivatives up to 30-40% of recovered metabolites (277).
Despite its genotoxic action in some test systems, caffeine does not bind
covalently to DNA from perfused liver of the rat (278). The inhibition of
post-replication repair of DNA has been suggested to be related to the induc-
tion of rat pituitary tumors by caffeine (228).
Quinine. Quinine is rapidly absorbed in the small intestine and is
largely metabolized in the liver. The metabolic products, many of them iden-
tified as hydroxy derivatives, are excreted in the urine and to a lesser
extent in the feces, bile and saliva (110).
Vinblastine and Vincristine. The metabolism of these two vinca alkaloids
in various animal species (171) and in humans (111) have been reviewed. They
are distributed to most tissues in rats and mice following parenteral admini-
stration. Appreciable amounts of these alkaloids are excreted unchanged,
314
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indicating that they are not metabolized to a great extent in rodents (279).
Metabolism of vinblastine and vincristine in humans involves only alteration
of side chains but not the ring system itself (280, 281). In humans (280) as
well as in dogs (282), one of the metabolic products of vinblastine is
deacetylvinblast ine.
5.3.2.3.2.5 ENVIRONMENTAL SIGNIFICANCE
For centuries, humans have been exposed to many of these alkaloids since
they occur in a wide variety of plants which serve as raw materials for the
preparation of a number of medicinal agents, edible oils, stimulating
beverages and tobacco products. The botanical sources and uses of these
alkaloids are presented in Table LVI. Long-term exposure to reserpine,
sanguinarine, caffeine or nicotine has been suspected to be the cause of
several human cancers.
Reserpine. The therapeutic uses in India of extracts of Rauwolfia
serpentia in the treatment of hypertension, insomnia, insanity and snake-bite
dates back many centuries. The root of the plant has long been known in
traditional Chinese medicine to have an antihypertensive effect. Reserpine,
the active agent of the plant, is still an important drug in modern medicine
for the treatment of hypertension and psychoses. It has been estimated that
millions of people in the United States have used reserpine. In 1974, reser-
pine was used in some 25% of all cases of diagnosed hypertension in the United
States and in 1976 in some 80% of cases in West Germany (see ref. 283).
The possible association between the use of reserpine and the development
of breast cancer in women has been a much debated subject. However, the epi-
demiological data from 14 case-control and 2 cohort studies, involving female
populations from various cities in the United States and Europe, do not lead
315
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Table LVI
The Botanical Sources and Uses of Some Plant Alkaloids3
Compound
Plants
Uses
Reserpine
Sanguinarine
Nicotine
Acronycine
Emetine
Caffeine
Quinine
Vinblastine,
Vincristine
Rauwolfia spp.
Argemone mexicana;
Chelidonium maius; etc.
Nicotina tabacum;
Duboisia hopwoodii
"Australia scrub ash"
Cephaelis impecacuanha;
C. acuminata
t »
Coffea spp.; Cola spp.;
Thea sinensis; Paulinia
spp.; Ilex paraguensis;
Theobroma Cacao
Cinchona officinalis;
C. succirubrum; C^.
calisaya; C. ledgeriana
Treatment of hypertension and
psychoses
A medicinal herb in India,
China, Africa, and West Indies;
adulteration of edible oil
A major constituent of tobacco;
agricultural insecticide
Experimental cancer chemotherapy
Treatment of amebic infections;
experimental cancer chemotherapy
An analgesic; preparation of
coffee, tea and other beverages
Treatment of malaria and noc-
turnal leg cramps, a bitter-
flavored constituent of some
carbonated beverages
Vinca rosea (Catharanthus Cancer chemotherapy
roseus); Catharanthus spp.
aSummarized from IARC Monographs Vols. 24 and 26, International Agency for
Research on Cancer, Lyon, France, 1980, 1981; A.G. Gilman, L.S. Goodman, and
A. Gilman (eds.), "The Pharmacological Basis of Therapeutics," MacMillan, New
York, 1980; The Merck Index, 10th ed. , Merck and Co., Rahway, N.Y., 1983.
See Table LI for structural formulas.
-------
to an unequivocal conclusion of relationship between reserpine use and breast
cancer. Although several studies found a positive relationship, there is only
a small increase in risk for long-term users (see 108, 172). This
apparent slight increase in risk may even be confounded by socio-economic
variables or other breast cancer risk factors ( cited In 283). Studies on
the effects of reserpine on prolactin level and on the incidence of breast
cancer in postmenopausal women suggest that slight increases in prolactin
level would not greatly increase the relative risk of breast cancer (284).
A case-control study (285) negates the hypothesis that reserpine exacer-
bates prostate cancer by stimulating serum prolactin production.
Sanguinarine. Sanguinarine has been found in Argemone mexicana (argemone
weed; yellow-flowered prickly poppy) and 49 other plant species belonging to
14 genera of the poppy-fumaria family (Papaveraceae). These plants grow
abundantly in both the tropical and the temperate regions of the globe. In
India, China, Africa and West Indies, argemone weed is used as a medicine.
People in these and other areas are also exposed to Sanguinarine through the
consumption of edible oils contaminated with argemone oil and/or the intake of
milk, liver and eggs from animals which fed on weeds producing Sanguinarine.
Hakim (161, 221) has noted a correlation between the geographical distribution
and density of sanguinarine-producing plants and the local incidences of
oesophageal cancer and stomach cancer. The high incidence of nasopharyngeal
cancer in some Chinese and Pillipinos populations is suspected to be related
to the smoking of opium which also contains Sanguinarine (221).
Nicotine. Nicotine is one of the alkaloids to which humans are most fre-
quently exposed, since it is a major constituent of tobacco Nicotiana tabacum
and Duboisia hopwoodii. The very strong statistical association of lung
316
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cancer and cigarette smoking is well established. It is also known that
tobacco ingredients are initiators and/or promoters of carcinogenesis.
Although the principal carcinogenic and co-carcinogenic agents in tobacco
smoke are polycyclic aromatic hydrocarbons (see ref. 286), it is possible that
nicotine may play the role of a cocarcinogen and/or promoter. The tobacco
specific nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosoamino)-l-(3-
pyridyl)-l-butanone, which have been found to be carcinogenic in rodents (see
Section 5.2.1.2, Volume IIIA), are derived from nicotine.
In the United States, a large quantity of nicotine sulfate was used as an
agricultural insecticide before being replaced by other chemicals.
^
Acronycine and Emetine. In experimental and clinical studies, both
alkaloids have been demonstrated to be effective agents against a broad
spectrum of tumors. Acronycine is obtained from the bark of the Australian
scrub ash whereas emetine is obtained from ipecac ("Brazil root"), the dried
root of Cephael is ipecacuanha or C_. acuminata (127, 224) . The emetine-
producing plants are native to Central America and Brazil but can also be
found in India and Malaysia. Since 1912 emetine is widely used for the treat-
ment of intestinal amebiasis, amebic hepatitis and other severe amebic infec-
tions (see 110) . A review in 1978 (1) estimated that 11-14 metric tons
of ipecac, which contains 12-14% of emetine, is imported into the United
States annually, mainly from Brazil.
Caffeine. Caffeine is a constituent of coffee, tea and other
beverages. It has a limited use in medicine, mainly as an analgesic and a
stimulant (107, 113). Most of the world population is exposed to caffeine to
a greater or lesser extent. In 1961, the Expert Panel of the Flavor and
Extract Manufacturers' Association of the United States considered caffeine to
317
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be GRAS ("Generally Regarded as Safe") for use in non-alcoholic beverages.
However, on October 15, 1980, the U.S. Food and Drug Administration proposed
that caffeine be no longer listed as GRAS, on the basis of more recent
research data (see 114).
A number of epidemiologic studies on the carcinogenic and teratogenic
effects of caffeine consumed in coffee and tea are available (revs. 114,
287). Stocks (288) compared the age-adjusted death rates due to cancer at
various sites and the annual consumption of coffee and tea in 20 countries
during 1964-1965. The author concluded that consumption of coffee positively
correlates with leukemia and cancer of the pancreas, prostate and ovary. A
i %
positive correlation also exists between the consumption of tea and cancer of
the intestine, larynx, lung and breast. However, Heyden (289) found several
flaws in this study. Cole (290) found an association between coffee-drinking
and bladder cancer in a case-control study. Later studies from the same
author (291) and from other investigators (292), however, failed to substan-
tiate the observed association. Shennan (293) reported a strong correlation
between coffee consumption and the rates of mortality from renal cancer in 16
countries. On the other hand, studies by others (294, 295) did not find such
association. More recently, MacMahon et al. (296) drew the attention to a
strong correlation between coffee consumption and pancreatic cancer in both
men and women. Review of the available data shows only ambiguous evidence for
reproductive or teratogenic effects due to ingestion of caffeine from coffee
(see 114).
Quinine. Quinine is the principal alkaloid of cinchona, the dried bark
of various Cinchona species. The history of this antimalarial agent dates
back for more than 300 years. In fact, quinine was the sole remedy for
malaria until World War II. This agent is still used for the management of
318
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malaria and for the relief of nocturnal leg cramps (110). It also finds use
as a bitter-flavored constituent of a widely used type of carbonated beverage
(230).
Vinblastine and Vincristine. In folk medicine, these two alkaloids from
the periwinkle plant (Vinca rosea) were used for controlling hemorrhage, for
the treatment of scurvy and toothache, and for healing chronic wounds. Since
the early 1960's, they have proved to be important agents, either singly or in
combination with other antineoplastic drugs, in the therapy of Hodgkins
disease, lymphosarcomas, choriocarcinomas, testicular tumors and other forms
of human neoplasms (1, 111, 297). Several case reports and epidemiologic
studies on the carcinogenic activity of these two drugs in humans have been
reviewed (171). In chemotherapy, with drug combinations including vincristine
or vinblastine, the two agents have been associated with the subsequent
development of leukemias.
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255. Govindwar, S.P., Kachole, M.S., and Pawar, S.S.: Food Chem. Toxicol.
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256. Ranadive, K.J., Gothoskar, S.V., and Tezabwala, B.U.: Int. J. Cancer
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260. Clemens, J.A., Shaar, C.J., Smastig, E.B., and Matsumoto, C. : Horm.
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261. Cazenave, J.-P., Reimers, H.-J., Perey, D.Y.E., and Mustard, J.F. :
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262. Lacassagne, A., Buu-Hoi, N.P., and Ba-Giao, N. : C.R. Acad. Sci.
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263. Faddejeva, M.D., Belyaeva, T.N., Novikov, J.P., and Shalabi, H.G.:
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264. Naiti, M., Nandi, R. , and Chaudhuri, K.: FEBS Lett. 142, 280 (1982).
265. Szuts, T., Olsson, S., Lindquist, N.G., and Ullberg, S.: Toxicology
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266. Stehlik, G., Kainzbauer, J., Tausch, H., and Richter, 0.: J.
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267. Oppelt, W.W., Zange, M., Ross, W.E., and Remmer , H.: Xenobiotica 5,
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268. Turner, D.M., Armitage, A.K., Briant, R.H., and Dollery, C.T.:
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269. Litterst, C.L., Mimnaugh, E.G., Reagan, R.L., and Gram, T.E.: Drug
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270. Hill, P., and Wynder, E.L.: Cancer Lett. 6, 251 (1979).
271. Nguyen, T.L., Gruenke, L.D., and Castagnoli, N. Jr.: J. Med. Chem. 22,
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272. Sullivan, H.R., Billings, R.E., Occolowitz, J.L., Boaz, H.E., Marshall,
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273. Tank-Liu, D.D., Williams, R.L., and Riegelman, S. : J. Phartnacol. Exp.
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274. Callahan, M.M., Robertson, R.S., Arnaud, M.J., Branfman, A.R., and
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275. Tank, B.K., Grant, D.M., and Kalow, W.: Drug Metab, Dispos. 11, 218
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276. Ferrero, J.L., and Neims, A.H.: Life Sci. 33, 1173 (1983).
277. Arnaud, M.J.: Biochem. Med. 16, 67 (1976).
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278. Szczawinska, K., Ginelli, E., Bartosek, I., Gambazza, C., and
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279. Castle, M.C., and Mead, J.A.R.: Biochem. Pharmacol. 27, 37 (1978).
280. Owellen, R.J., Hartke, C.A., and Hains, F.O.: Cancer Res. 37, 2597
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281. Bender, R.A., Castle, M.C., Margileth, D.A., and Oliverio, V.T.: Clin.
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282. Creasey, W.A., and Marsh, J.C.: Proc. Am. Assoc. Cancer Res. 14, 57
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283. Labarthe, D.R., and O'Fallon, W.M. : J. Am. Med. Assoc. 243, 2304
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284. Ross, R.K., Paganini-Hill, A., Krailo, M.D., Gerkins, V.R., Henderson,
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285. Newball, H.H., and Byar, D.P.: Urology 2, 525 (1973).
286. Weisburger, J.H., Cohen, L.A., and Wynder, E.L.: On the Etiology and
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337
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287. Miller, A.B.: Coffee and Cancer. In "Carcinogens and Mutagens in the
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288. Stocks, P.: Br. J. Cancer 24, 215 (1970).
289. Heyden, S. : 2. Ernahrungswiss. 14, 11 (1972).
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i ^ """"""
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SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.3.2.3
1. Bull, L.B., Culvenor, C.C.J., and Dick, A.T.: "The Pyrrolizidine
Alkaloids." John Wiley and Sons, New York, 1968, 293 pp.
2. McLean, E.K.: Pharmacological Reviews 22. 429-483 (1970).
3. International Agency for Research on Cancer: "Some Naturally Occurring
Substances," LARC Monographs on the Evaluation of Carcinogenic Risk of
Chemicals to Man, Vol. 10. Int. Agency Res. Cancer, Lyon, France,
1976, pp. 265-342.
338
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4. International Agency for Research on Cancer: "Some Pharmaceutical
Drugs," IARC Monographs on the Evaluation of Carcinogenic Risk of
Chemicals to Man, Vol. 24. Int. Agency Res. Cancer, Lyon, France,
1980, pp. 211-241.
5. International Agency for Research on Cancer: "Some Antineoplastic and
Immunosuppressive Agents," IARC Monographs on the Evaluation of
Carcinogenic Risk of Chemicals to Man, Vol. 26. Int. Agency Res.
Cancer, Lyon, France, 1981, pp. 349-384.
6. Schoental, R.: Carcinogens in Plants and Microorganisms. In "Chemical
Carcinogens" (C.E. Searle, ed.), ACS Monograph 173, American Chemical
i- ^
Society, Washington, D.C., 1976, pp. 626-689.
7. Culvenor, C.C.J., and Jago, M.V.: Carcinogenic Plant Products and
DNA. In "Chemical Carcinogens and DNA" (P.L. Grover, ed.), CRC Press,
Boca Raton, Florida, 1979, pp. 161-186.
8. Miller, A.B.: Coffee and Cancer. In "Carcinogens and Mutagens in the
Environment" (H.F. Stich, ed.), Vol. Ill, CRC Press, Boca Raton,
Florida, 1983, pp. 13-20.
9. Timson, J.: Mutat. Res. 47, 1-52 (1977).
10. Thayer, P.S., and Palm, P.E. : CRC Grit. Rev. Toxicol. _3_, 345-369
(1975).
11. Oser, B.L., and Ford, R.A.: Drug Chem. Toxicol. 4. 311-329 (1981).
12. Hakim, S.A.E.: Maharashtra Med. J. 17, 109-130 (1970).
339
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