CURRENT AWARENESS DOCUMENT
TETRAHYDROPYRIDINE DERIVATIVES, TANNINS. FLAVONOIDS, AND VARIOUS
STRUCTURAL TYPES OF INDUSTRIALLY-USED LIPID CHEMICALS
AND OTHER SUBSTANCES OF PLANT ORIGIN
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.
Extradivisional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5.3.2.6 Miscellaneous Plant Carcinogens
5.3.2.6.1 Betel Nut Carcinogens
5.3.2.6.1.1 INTRODUCTION
As early as 600 A.D., the chewing of betel quid with or without tobacco
and other spicy .ingredients, had been documented as a common practice in the
Orient. Betel quid consists of betel nut (the seeds of the betel palm Areca
catechu L.) wrapped in the leaf of the betel vine (Piper betie L.), together
with lime (calcium hydroxide). The role of lime is to neutralize the acidity
and astringent taste of betel nut, whereas the betel vine leaf is a carmina-
tive and "sweetens" the breath"*. The stimulating and narcotic effects of betel
quid are, however, due largely to the betel nut alkaloids. At least six
reduced pyridine alkaloids are present in betel nut and of these, arecoline,
and its hydrolyzed products arecaidine, guvacoline and guvacine (see Table
LXXV) have received the most attention. Laboratory studies have shown that
arecoline is both mutagenic and carcinogenic. Arecaidine also displays muta-
genic, clastogenic and cell-transforming activities. Furthermore, carcino-
genic nitrosamines are suspected to be formed from arecoline during chewing,
since human saliva contains nitrite. Besides alkaloids, betel nut contains
other carcinogens such as tannins (see Section 5.3.2.6.2). Several flavonoid
compounds found in betel nut, such as (+)-catechin, cyanidin and delphinidin
possess appreciable clastogenic activity (see Section 5.3.2.6.3.3). On the
other hand, eugenol (see Section 5.2.1.4, Vol. IIIB) and several flavonoid
glycosides, for example, apigenin and luteolin (see Section 5.3.2.6.3), all
present in betel leaf, are devoid of mutagenic activity. These findings are
consistent with the observation that extracts of betel quid (i.e., betel nut,
betel leaf and lime) and betel nut, but not that of betel leaf alone, induce
444
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mutation and tumors in various test systems. Interestingly, the tumorigenic
activity of betel quid and betel nut are enhanced when they are administered
in combination with tobacco.
For many years, the possible connection between the occurrence of oral
and oesophageal cancer and the use of betel quid has attracted the attention
of oncologists. At present, a number of epidemiclogic studies have correlated
the high incidence of oral cancer with the habit of chewing betel quid in
several areas of the world.
5.3.2.6.1.2 PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL EFFECTS
5.3.2.6.1.2.1 Physical and 'cftemical Properties. The composition and
chemistry of betel nut and betel leaf have been reviewed (1, 2). There is a
general belief that much of the pharmacological and toxicological properties
are attributed to the alkaloids in betel nut, particularly to arecoline and
arecaidine. Betel nut contains about 0.07-0.50% arecoline, a small amount of
arecaidine and traces of guvacoline and guvacine. These alkaloids are all
derivatives of 1,2,5,6-tetrahydropyridine and contain a 3,4-double bond in
their molecules (see Table LXXV). Arecoline (1,2,5,6-tetrahydro-l-methyl-3-
pyridinecarboxylic acid methyl ester), is an oily liquid with a boiling point
o
of 209 C. It is soluble in chloroform and miscible with water, ethanol and
ether (3). Upon chemical or enzymic hydrolysis, arecoline looses its methyl
ester grouping and yields the corresponding acid, arecaidine, which is a solid
soluble in water and insoluble in ethanol, chloroform, ether or benzene (3).
In neutral aqueous solution at 37°C or in boiling ethanolic solutions,
arecoline readily S-alkylates N-acetyl-L-cysteine or glutathione; arecaidine
also reacts with thio groups at the 3,4-double bond but the reaction is
slower. Reaction of aqueous solutions of arecoline at pH 3-5 with an excess
445
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CH3
cotlivereste
orCa(OH)2
N
rx' ^ rotliveresterose
*COOCH3
Arecoline, / Arecaidine
TOOH |
S-CH2-CH-COOH |
i "HTCH3 i
* N-Acetyl-S-(3-carboxy- Q T
-l-methyl-piperid-4-yl)
H -L-cysteine H
COOCH, COOH
Guvocoline Guvacine
Table LXXV
in the
-------
of sodium nitrite yields the nitrosation products N-nitrosoguvacoline,
3-(methylnitrosamine)-propionitrile and 3-(methylnitrosamino)-propionaldehyde
(4) (see Fig. 18).
5.3.2.6.1.2.2 Biological Effects Other Than Carcinogenicity
Pharmacological Effects. The discussion of the biological effects of
betel quid would be incomplete without mentioning its pharmacological proper-
ties which is the basis of addiction of millions of individuals in the Orient,
principally on the Indian subcontinent, in China and Southeast Asia. The
chewing of betel quid is said to promote salivation, sweeten the breath,
strengthen the gums, improve appetite and taste, and produce stimulating and
exhilarating effects on the system (1, 2). Studies on the constituents of
betel quid established that these effects are due primarily to arecoline, the
major betel nut alkaloid. Arecoline is cholinergic and like acetylcholine and
pilocarpine, has a stimulating effect on the parasympathetic system and an
inhibiting effect on the sympathetic system. Arecoline stimulates salivary
secretion, causes a lowering of blood pressure due to vasodilation, reduces
the sugar and lipid content of the blood and increases urine flow and intes-
tinal peristalsis. Structure-activity relationship analysis shows that the
methyl ester group of arecoline is critical for the parasympathetic action.
Replacement of the methyl ester grouping by an ethyl ester increases, whereas
by a propyl or butyl ester markedly decreases, the hypotensive effect of
arecoline in the cat. Hydrogenation of the double bond also leads to a reduc-
tion of the hypotensive effect (cited in 2). Differences in pharmacological
effects among arecoline, arecaidine and analogues are probably related to
their different rates of reaction with thiol enzymes. Some of the psychogenic
effects of betel quid chewing may result from the inhibition by arecaidine and
guvacine of the uptake of |-aminobutyric acid, an inhibitor of synaptic
transmission in brain tissue (5).
446
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CH3
r i
^Af pH3-5
XOOCH3
Arecoline
NO
XOOCHj
N-Nitrosoguvacoline
CH3
M
NO
^CN
3-(Methylnitrcsamino)-
-propionitrile
CH,
3-(Methynitrosamino)-
-propionaldehyde
Fig. 18. N-Nitrosation of arecoline.
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Toxic Effects. A combination of undesirable effects including red and
congested face, sensation of heat in the head, nausea and giddiness are common
to beginners in betel quid chewing. Frequent chewers may experience numbness
at the mouth, dizziness, loose motions and strong intestinal irritations
(2). Most of these effects are due to arecoline present in betel nut which is
lethal to mice at 100 mg/kg body weight after s.c. administration (3). The
LDcQ value of arecoline hydrochloride in mice by i.p. injections is 154 mg/kg
(6). When injected i.p. into Swiss mice, a polyphenolic fraction of betel nut
was found to decrease glycogen and increase sialic acid in the lung and kidney
tissues. This fraction also decreased nucleic acid and protein content in
t •%
almost all tissues examined (7).
Mutagenic Effects. Several short-term mutagenicity assays have demon-
strated the presence of numerous mutagens in betel nut or betel quid.
Aqueous extracts of betel nut or betel quid are mutagenic in strains
TA100 and TA1535 of Salmonella typhimurium; addition of S-9 mix to the test
system enhances the mutagenicity (8). These extracts also cause a significant
increase in the induction of micronuclei in the erythrocytes of mice (9).
This supports the observations that individuals who habitually chew betel nut
or betel quid have a higher frequency of micronucleated cells in the buccal
mucosa (10). Clastogenic acivity has been reported in the saliva of volun-
teers chewing betel nut or betel quid (11). Furthermore, extracts of betel
nut induce point mutations in V79 Chinese hamster cells in vitro (9). Betel
leaf extracts, on the other hand, exhibit negative results in the Ames test
(8), in the micronucleus test (9), and in the sister-chromatid exchange (12)
and the Drosophila (13) assays. Nonetheless, chromosome-damage has been
reported in human leukocyte cultures treated with betel leaf extracts (14) and
with the saliva of individuals who chew betel leaf (11). Several flavonoids
447
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present in betel leaf are free of clastogenic properties (see Section
5.3.2.6.2). Furthermore, betel leaf contains antioxidants which are known to
be anticarcinogenic and antimutagenic (15). The addition of betel leaf
extracts has been shown to markedly decrease the mutagenic effects of betel
nut (8). On the other hand, the mutagenicity of betel nut or betel quid is
potentiated by tobacco, suggesting that the betel mutagens and tobacco consti-
tuent^) may act synergistically (8, 9).
A number of mutagenic agents have been identified in betel nut. Areco-
line and arecaidine display a dose-dependent increase in mutagenicity when
studied in four tester strains of Salmonella typhimurium, with strain TA100
i > " ' ~
being the most sensitive. Arecoline is mutagenic even without metabolic acti-
vation; when tested in the presence of S-9 mix, however, the mutagenicity is
enhanced (8). Shirname and coworkers (9) found arecoline positive in a micro-
nucleus test and in inducing a significant increase in mutation frequency of
V79 Chinese hamster cells. The induction of sister-chromatid exchange (SCE)
by arecoline and arecaidine has been demonstated in mouse bone marrow cells in
vivo (16, 17). Concomitant treatment of animals with caffeine and arecoline
results in an additive effect on the SCE frequencies (16). Arecoline produces
chromosomal aberrations in different test systems (18, 19). A potentiating
effect on clastogenicity was noted when eugenol (from betel leaf), chlorogenic
acid (from tobacco), quercetin (see Section 5.3.2.6.3) or the transition metal
Mn + was added concurrently with arecoline to Chinese hamster ovary (CHO)
cells (19). Apart from betel nut alkaloids, the tannic acid fraction of betel
.nuts induces chromatid breaks and exchange in mammalian cells and gene conver-
sion in yeast (20). (+)-Catechin and cyanidin (see Section 5.3.2.6.3), two
polyphenolic compounds in betel nut, display strong clastogenic effects in
Chinese hamster ovary cells (20).
448
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5.3.2.6.1.3 CARCINOGENICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
5.3.2.6.1.3.1 Overview. Considerable work has been carried out to examine
the carcinogenic properties of betel nut, betel leaf and betel quid in experi-
mental animals. The results of some key carcinogenesis studies by local or
oral administration of the extracted materials to animals are summarized in
Table LXXVI. The data of these studies suggest that betel nut and betel quid
(the combination of betel nut, betel leaf and lime), but not betel leaf, are
carcinogenic; the carcinogenic effects of betel quid are mainly due to the
alkaloids in the nut, particularly to arecoline. In accord with the muta-
genicity data, ingredients in tobacco enhance whereas those in betel leaf
inhibit the carcinogenic action of betel nut. 3-(Methylnitrosamino)-propio-
nitrile, one of the nitroso compounds suspected to be formed in the oral
cavity of betel quid chewers, has been shown to be strongly carcinogenic in
the rat.
5.3.2.6.1.3.2 Carcinogenicity of Betel Quid, Betel Nut, and Betel Leaf.
Early studies have demonstrated the induction of low incidence of papillomas
and squamous cell carcinomas in mice painted on the ears with aqueous extracts
of betel quid and tobacco (1) or instilled into the vagina (21).
Although no malignant tumors were produced, inflammatory lesions accompanied
by hyperplasia were induced in the buccal pouch of hamsters treated with
beeswax pellets that contained various ingredients of betel quid (32). In
1971, Suri _et_ al. (25) succeeded in inducing tumors in 8 of 19 (38%) Syrian
golden hamsters treated topically with dimethylsulfoxide (DMSO) extracts of
betel nut to the mucosa of the buccal pouch. Subsequently, Ranadive and his
colleagues (22, 23) reported the emergence of transplantable fibrosarcomas at
the injections site of 30-60% of Swiss mice administered aqueous extracts of
betel nut or betel quid subcutaneously. The polyphenol fraction of betel nut,
449
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Table LXXVI
Carcinogenicity of Betel Quid, Betel Nut Betel Leaf,
Arecoline and Related Compounds
p.l of 2
Substance
Betel quid3
and tobacco
Betel quida
Betel nut
Betel nutb and
betel leafc
Betel leafc
Arecoline
Species and
strain
Mouse, Swiss
Mouse, Swiss
Mouse, Swiss
Mouse, Swiss
Hamster, Syrian
golden
r >
Mouse, Swiss
Mouse, Swiss
Mouse, C17
Mouse, Swiss
Rat, NIH Black
Rat, AC I
Mouse, Swiss
Mouse, Swiss
or C17
Rat, ACI
Hamster, Syrian
golden
Hamster, Syrian
golden
Mouse, Swiss
Mouse, Swiss
Route
topical
s .c .
oral
i.p.
topical
s .c .
oral
oral
i.p.
s .c .
oral
s .c .
oral
oral
topical
oral
oral
s.c. or
i.p.
Principal organ
affected
Ear, vagina
Local sarcoma
Lung
None
Buccal pouch
Local sarcoma
Multiple sites
Multiple sites
None
Local sarcoma
Urinary bladder ,
hematopoietic
tissue
None
None
None
Buccal pouch,
esophagus
None
Liver, lung,
and stomach
None
Reference
(1,
(22,
(8,
(23)
(22,
(22,
(8,
(24)
(23)
(26)
(27)
(22,
(8,
(27)
(28)
(28)
(29)
(23)
21)
23)
24)
25)
23)
24)
23)
24)
-------
Table LXXVI (continued)
p.2 of 2
Substance
3-(Hethyl-
Species and
strain
Rat, F344
Route
s .c .
Principal organ
affected
Gastrointes-
Reference
(30)
nitrosamino)-
propionitrile
tinal system,
nose and tongue
N-Nitroso-
guvacoline6
Rat , Sprague-
Dawley
oral
None
(31)
aConsists of betel nut, betel leaf and lime (calcium hydroxide).
Seed of the betel palm, Areca catechu L.
cLeaf of the betel vine, Piper betle L.
See Table LXXV for structural formula.
eSee Figure 18 for structural^formulas.
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which contains a high content of tannin, induced 100% tumors in treated mice
(23). Interestingly, no neoplasms developed in mice receiving aqueous
extracts or a polyphenolic fraction of betel nut or aqueous extracts of betel
quid, by i.p. injections (23). On the other hand, significant incidences of
tumors in the liver, lung and stomach were found in mice of strains Swiss and
C17 fed intragastrically once daily, 5 times a week, 0.1 ml aqueous extract of
betel nut (8, 24). Among 18 Swiss mice fed the polyphenol fraction of betel
nut, one developed hemangioma of the liver, and 2 had salivary gland tumors
(24). Intragastric administration of aqueous extracts of betel quid elicited
lung tumors in 26% of Swiss mice. Betel leaf extracts were not carcinogenic
! ^
in mice under similar study conditions (8, 24).
The carcinogenic activity of betel nut has also been tested in the rat.
Aqueous extracts of betel nut were injected s.c. into the flank of 15 male and
15 female outbred NIH black rats once a week. After 56 weeks of treatment,
all 30 rats developed local tumors at the injection site (26). A low inci-
dence of transitional cell carcinoma of the urinary bladder and myeloid
leukemia was observed in groups of inbred strain ACI rats fed a diet contain-
ing 20% of dry powder of betel nut, with or without 1% calcium hydroxide
(lime), for up to 480 days. The forestomach papilloma found in one animal
among rats given betel leaf diet was not considered to be due to the admini-
stration of betel leaf (27).
5.3.2.6.1.3.3 Carcinogenicity of Arecoline and Related Compounds. The
ability of arecoline and arecaidine to alkylate thio-compounds prompted Ashby,
Styles and Boyland (33) to evaluate the carcinogenic potential of these two
betel nut alkaloids in an in vitro cell-transformation assay. Both compounds
exhibited an almost identical positive response in the assay. In a first
study Shivapurkar ££._al_. (23) was unsuccessful to induce tumors in Swiss mice
450
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injected subcutaneously or intraperitoneally with arecoline. However, a Later
study by the same group of workers (29), administering arecoline by gavage to
groups of male and female Swiss mice at a dose of 1 ing/day/mouse five times
weekly, showed that arecoline is carcinogenic in mice, inducing tumors in the
liver, lung and stomach. Dunham et al. (28) reported the development of
esophageal papillomas in 2 of 9 Syrian golden hamsters following application
of arecoline (in water) and calcium hydroxide (lime) into the cheek pouch.
Hamsters that had arecoline in dimethylsulfoxide painted on the pouch, or
those that ingested arecoline in the diet (0.1%), did not develop tumors in
the pouch or the esophagus (28).
' >
Attempting to support the hypothesis that the carcinogenic action of
betel quid or betel nut may be due to nitrosamine(s) formed during the chewing
of betel nut, 3-(methylnitrosamino)propionitrile and N-nitrosoguvacoline, two
of the in vitro nitrosation products of arecoline, were tested for carcino-
genic activity. Upon s.c. injection of 2.13 mg 3-(methylnitrosamino)-propio-
nitrile 3 times/week for 20 weeks, all of the 15 male and 15 female F344 rats
developed tumors within 24 weeks. The tumors produced were papillomas and
carcinomas of esophagus (90% incidence), nasal cavity (70% incidence) and
tongue (37%), and carcinomas of pharynx (7%) and papillomas of forestomach
(7%); no tumors were seen in the controls (30). The potent carcinogenic
activity of this compound is not too surprising since its lower homologue,
2-(methylnitrosamino)-acetonitrile was already known to be a carcinogen (see
Section 5.2.1.2.3.2.1, Vol. IIIA). On the other hand, N-nitrosoguvacoline,
administered to 15 male or female 8-10 weeks old Sprague-Dawley rats in drink-
ing water (at 0.88 mM, total dose is 750 mg), 5 days/week for up to 50 weeks,
did not induce a significant incidence of tumors (31).
451
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5.3.2.6.1.3.4 Modification of Carcinogenesis. Since betel quid and the com-
ponents of the "chew" contain an array of chemicals, they will expectably
interact in bringing about the observed carcinogenic response. It has been
hypothesized that tobacco may contain substances that potentiate the carcino-
genicity of betel nuts. Indeed, several investigators (e.g., 22, 25) have
noted the enhancing effect of tobacco on betel nut carcinogenesis in the
hamster cheek pouch as well as on the mouse skin. In 1964, Atkinson et al.
(34) drew attention to the possible role of lime (calcium hydroxide) in the
carcinogenic activity of the betel quid combination. Mori et_ al_. (27) demon-
strated later that the incidence of epidermal hyperplasia on the tongue, oral
I ^
mucosa or forestomach of rats fed a betel nut diet mixed with lime was signi-
ficantly higher than that of animals given betel nut diet alone. Betel leaf,
on the other hand, has been repeatedly shown to exhibit a protective effect
against the carcinogenicity of betel nut in mice (8, 23, 24). Bhide et al.
(29) noted a modifying role of vitamin B deficiency in arecoline tumorigenesis
in the mouse. Female Swiss mice administered arecoline by gavage, or male
Swiss mice similarly treated with a mixture of arecoline, KNOo and lime,
developed tumors only when they were kept on a vitamin B-deficient diet but
not on a normal diet.
Consistent with the carcinogenic activity of betel nut and the inhibitory
effect of betel leaf, carcinogenesis of benzol a]pyrene in the buccal pouch of
hamsters is enhanced by betel nut and decreased by betel leaf (35).
5.3.2.6.1.4 METABOLISM AND MECHANIC OF ACTION
In the rat, arecoline is metabolized to arecaidine (36). Arecoline is
also converted to arecaidine by rat liver homogenates or by reaction with
lime, a component of the betel quid (37). Administration of arecoline or
452
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arecaidine to rats resulted in the same urinary metabolite N-acetyl-S-(3-
carboxy-l-methylpiperid-4-yl)-L-cysteine (36) (see Table LXXV).
Although a number of carcinogenic and genotoxic compounds, tannins,
flavonoids and phenolics (see Section 5.3.2.6.2 and Section 5.3.2.6.3) have
been isolated from betel mixtures in recent years, the notion prevails that
the betel alkaloids arecoline and arecaidine are the carcinogenic principles
of betel quid. This view is based primarily on the alkylating activity of the
two alkaloids. Both in vivo and in vitro studies (36, 38) have shown that the
3,4-double bond of arecoline and arecaidine react with thio-containing com-
pounds. The addition of the thiols to the 3,4-double bond of these alkaloids
could occur by a nucleophilic addition involving attack by thiol on the acti-
vated ethylenic bond (36). Formation of arecaidine-3,4-epoxide as a reactive
intermediate is another possible mechanism which has been proposed (17).
CH3
"COOH
Arecaidine Arecaidine-3,4-epoxide
However, in vitro studies (36) did not detect any reaction between arecoline
or arecaidine and nucleic acid bases. Whether such interaction occurs in vivo
is still unknown at the time of this writing. Chemically, being an
o<,/3-unsaturated carboxylic acid, arecaidine may react with cellular nucleo-
philes by the Michael-type addition similar to that with acrylic acid (see
Appendix I).
The putative role of 3-(methylnitrosamino)propionitrile, the N-nitrosa-
tion product of arecoline, in carcinogenesis in chewers of betel quid warrants
further investigation. This compound most likely acts via a mechanism similar
to that of its lower homologue, 2-(methylnitrosamino)acetonitrile, discussed
in Section 5.2.1.2.3.2.1, Vol. IIIA.
453
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5.3.2.6.1.5 ENVIRONMENTAL SIGNIFICANCE
Areca catechu L., a tall and slender palm tree, is cultivated widely in
South Pacific islands and tropical countries in Southeast Asia such as India,
Sri Lanka, Malaysia, Singapore, Indonesia, Burma, Thailand, Philippines,
Taiwan and China. The use of its seeds, called betel nut, areca nut, or
"pinnon" (in Chinese) as masticatory and local medicine in India and China
dates back to about 600 A.D. or earlier. In the ancient systems of Indian
Ayurvedic and Chinese medicine, betel nut was/is used for the treatment of
urinary disorders, bleeding gums, ulcers, vaginal discharges and heartburn in
pregnancy. It has also been used as an anthelminthic remedy against tapeworms
and roundworms (2). The importance of betel nut, however, is due to its wide
use for addictive chewing in India, Sri Lanka, Malaysia and other countries in
the Far East. According to current estimates over 250 million individuals in
Southeast Asia are addicted to chewing betel quid (see 10). Coinciding with
this, there is an extraordinarily high incidence of oral cancer in regions of
high prevalence of the habit/addiction of betel chewing. Epidemiological
studies provide considerable evidence to indict betel and/or tobacco chewing
as important factors in the etiology of oral, pharyngeal and esophageal
cancers in these regions (rev. in 1, 20, 39, 40). The high risk of esophageal
cancer in Bombay, India (41) and the high incidence of oral cancer in New
Guinea (34) are believed to be due solely to betel nut since tobacco is
usually not included in the "chew."
5.3.2.6.2 Plant Phenolics: Tannins and Related Compounds
5.3.2.6.2.1 INTRODUCTION
Phenolic compounds have a wide, if not universal, occurrence in the plant
kingdom. It has been estimated that over 2,400 phenolic aglycones are known
454
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to be present in plants (42). They are extremely useful in chemotaxonomic
differentiation of plant species, because the qualitative diversity of
phenolics is roughly proportional to the phylogenetic development of the
plants. Most phenolic compounds of toxicological interest are found in angio-
sperms in which they appear to play a protective role in response to physical
injury, microbial infection or infestation and against consumption by herbi-
vores (43). A number of plant phenolics have been found to be weak to
moderately active carcinogens. This section focuses on tannins and related
compounds. Other phenolic compounds such as flavonoids (Section 5.3.2.6.3),
rotenone (Section 5.3.2.6.4.4), capsaicin (Section 5.3.2.6.4.5), cannabinoids
r •>
(Section 5.3.2.6.4.6) and simple phenolics (Vol. IIIB, Section 5.2.2.5) are
discussed in other sections of this series of monographs.
Tannins are polyphenolic compounds with molecular weights ranging from
500 to 3,000. Tannic acid,* a hydrolyzable form of tannin, was extensively
used between 1925 and 1942 and during World War II in burn treatment due to
its astrigent effect. In 1943, Korpassy and associates (see 44) found severe
liver necrosis in tannic acid-treated burn victims and raised suspicion that
tannic acid may be hepatocarcinogenic. The suspicion was substantiated by
animal data which indicated the induction of liver tumors in rats given s.c.
injections (but not skin painting) of tannic acid (45). Subsequently, Kirby
(46) showed that tannins present in various plant extracts are also carcino-
genic. Since 1970, a series of epidemiologic surveys by Morton (47-51) and a
variety of other investigators (see Section 5.3.2.6.2.5) have revealed a
*The term "tannic acid" has often been incorrectly used as a general terra for
tannins. Tannic acid should only refer to the hydrolyzable type of tannins of
the gallotannin class.
455
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strong correlation between high incidence of esophageal cancer in various
geographic areas throughout the world and the consumption of tannin-rich
herbal teas or medicines by their inhabitants. A variety of plants from these
areas has been tested and found to be carcinogenic by s.c. injections but not
by oral administration; in most cases, the carcinogenic activity is indeed
associated with the tannin fraction of the plant extracts.
5.3.2.6.2.2 PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL EFFECTS
5.3.2.6.2.2.1 Physical and Chemical Properties
The physical and chemical properties of tannins have been described in a
i >
number of reviews (42, 52-54). Some representative structural formulas of
tannic acid, condensed tannins, their components and related compounds are
depicted in Table LXXVII. Tannins are complex polyphenolic esters of sugar or
polyhydric alcohol or polymeric flavonoids with molecular weight ranging from
500 to 3,000. Tannins are generally classified into two types — "hydrolyz-
able tannins" and "condensed tannins." Hydrolyzable tannins may be further
subdivided into gallotannins and ellagitannins, which, upon heating with
mineral acids, yield phenolic acids (gallic acid and ellagic acid, respec-
tively), glucose and, in some cases (e.g., tara tannin), quinic acid. The
commercially available tannic acid consists mainly of gallotannic acid (gallo-
.tannin) with 6-9 units of gallic acid esterified to glucose. The condensed
tannins (catechin tannins) are polymeric flavonoids composed predominantly of
leucoanthocyanidin units. They do not readily break down under physiological
conditions but may release flavonoid monomers (e.g., catechin) after drastic
treatment.
Depending on the size and the type of compound, tannins may be quite
water soluble. One gram of tannic acid dissolves in 0.35 ml water or 1 ml
456
-------
Structural Formulas
CH2_(,Tvdigallate)
O
(R=galloyl or
m . digalloyl)
HO
HO
COOH
HO
Gallic acid
HO
HO OH
HO
COO
HO
COOH
m-Digallic acid
Table
of Tannins and Related Conpounds
HO
7
.O
HOOC
Chlorogenic acid
(R=caffeyl)
Quinic acid IR =
= CH-COOH
Caffeic acid IR=H)
Ferulicacid (R=CH3)
OH OH
OH OH
Condensed tannin
Catechin (R = H)
Leucoanthocyanidin (R = OH)
-------
warm glycerol. Tannic acid is also soluble in alcohol and acetone but is
practically insoluble inmost organic solvents (53). The solubility of
tannins decreases with an increase in molecular weight. Condensed tannins
with molecular weight greater than 5,000 are very poorly soluble in aqueous
solutions and are practically inert physiologically (42). Tannic acid
gradually darkens on exposure to air and light. It decomposes when heated to
210-215°C, yielding mainly pyrogallol and carbon dioxide. High molecular
weight condensed tannins undergo further polymerization and partial oxidation
when heated, forming inert, insoluble, poorly characterized "phlobaphenes"
(42). Owing to the presence of multiple hydroxyl groups, tannins are noted
for their ability to bind to proteins forming crosslinks. Insoluble precipi-
tates are formed when tannic acid is mixed with albumin, starch or gelatin.
Oxidation of tannins decreases the protein-binding activity because of conver-
sion of hydrogen bond donor hydroxyls into acceptor quinone carbonyls. The
protective effect of calcium hydroxide against the toxicity of tannins (e.g.,
55, 56) has been attributed to the acceleration of phenol oxidation by
alkaline pH. Tannins can also be precipitated by alkaloids such as caffeine
and quinine; these alkaloids have been used to separate tannins from plant
extracts (57).
Chlorogenic acid (3-caffeoylquinic acid) has a melting point of 208°C, an
optical rotation ([<*]D) of -35.2° at 26°C and a pKa of 2.66 at 27°C. It is
quite soluble in water (4% at 25°C) and is freely soluble in alcohol and
acetone. Upon heating with dilute hydrochloric acid, chlorogenic acid breaks
down to its components, quinic acid and caffeic acid. It forms a black com-
pound with iron, believed to be responsible for the darkening of cut potatoes
(3). Under mildly acidic conditions, chlorogenic acid is a powerful catalyst
for N-nitrosation of piperidine by sodium nitrite (58).
457
-------
5.3.2.6.2.2.2 Biological Effects Other Than Carcinogenicity
Toxic ity. The toxic effects of tannins and related phenolic compounds
have been thoroughly reviewed in 1981 by Singleton (42). Tannins from various
sources are of relatively low toxicity when administered orally but are
considerably more toxic when given parenterally. The reported oral LDjQ
values of tannic acid are 3.5, 2.3 and 5 g/kg body weight for mice (59), rats
(60) and rabbits (61), respectively. The LD^Q of tannic acid in mice is only
about 0.04 g/kg body weight by i.v. injection (59). The oral LDcQ of a
hydrolyzable tannin (of the gallotannin class), isolated from Quercus havardi
(shin oak), in rabbits is 6.,9>g/kg body weight (62). The s.c. LD^Q values in
mice range from 0.1 g/kg body weight for hydrolyzable tannins (from myrobalans
and chestnut) to 1.6 g/kg for condensed tannins (from quebracho and mimosa)
(63). The i.p. LDcQ of a tannin, isolated from bracken fern, in mice is 0.16
g/kg body weight (64). Gallic acid (oral LDcg, 5 g/kg in rabbits) has approx-
imately the same degree of acute toxicity as tannic acid in rabbits (61).
Chlorogenic acid and caffeic acid are also relatively nontoxic; their i.p.
LDcjQ values in rats are greater than 2.4 and 1.25 g/kg body weight, respec-
tively (65) .
Tannins (especially tannic acid) were used between 1925 and the end of
World War II in burn treatment (rev. in 42, 44, 54). Liver and kidney
toxicity and occasional fatalities were observed. Since gallic acid had no
such toxic effects, absorption of tannins into the bloodstream must have
occurred. Condensed tannins were somewhat less toxic than hydrolyzable
tannins in burn treatment. Between 1946 and 1964, tannic acid was used along
with barium enemas to improve X-ray diagnosis of colitis (rev. in 66). Severe
acute liver damages, and at least eight fatalities, were produced in a number
of these patients.
458
-------
Mutagenicity. There is little information available on the mutagenicity
of tannins. A tannin (condensed type) fraction isolated from bracken fern
(Pteridium aquilinum) is nonmutagenic in the Ames Salmonella test (67).
Tannic acid is also nonmutagenic toward Escherichia coli B/r WP2 (68).
Chlorogenic acid and its components, caffeic acid, and quinic acid, are all
nonmutagenic in the standard Ames test (69, 70). However, when assayed in the
presence of the transition metal, Mn++, chlorogenic acid and caffeic acid
display some weak mutagenic activity (70). Chromosome aberration tests (19,
71, 72) using Chinese hamster ovary (CHO) cells indicated that tannic acid and
tannins from a variety of origins (e.g., apple juice, grape juice and wine),
i- >
gallic acid, chlorogenic acid and caffeic acid are all clastogenic, inducing
significant increases in chromosome aberrations, chromatid breaks and
exchanges. The clastogenic activity of these phenolic compounds can be sub-
stantially increased by the inclusion of some transition metals, such as Mn ,
Cu"1"*" (but not Fe"1"1"). Synergism between chlorogenic acid and arecoline (a
pyridine alkaloid present in betel nut, see Section 5.3.2.6.1) has also been
observed. Combination of chlorogenic acid and arecoline with Mn++ further
increases the clastogenic activity. There is some evidence that, like the
polyhydric phenols (see Vol. IIIB, Section 5.2.2.5.2.2), caffeic acid and
chlorogenic acid may owe their mutagenic and clastogenic activities to the
presence of the two aromatic hydroxy groups. Methylation of the 3-hydroxy
group of caffeic acid yields a completely nonclastogenic compound, ferulic
acid (3-methoxy-4-hydroxycinnamic acid). It is possible that a semiquinone-
type reactive intermediate (seg Vol. IIIB, Section 5.2.2.5.2.1) may be
involved in the clastogenic action of caffeic and chlorogenic acid.
A number of tannins and related compounds have been shown to be modu-
lators of other mutagens. Shimoi et %1. (68) showed that tannic acid signi-
459
-------
ficantly suppresses the UV- and 4-nitroquinoline-induced mutagenesis in E.
coli but has no modulating effect on X-rays or N-methyl-N'-nitro-N-nitroso-
guanidine. The antimutagenic effects of tannic acid can be observed only in
excision repair-proficient strains suggesting that the mode of action of
tannic acid may involve enhancement of excision repair probably by activating
the repair enzymes or by interacting with DNA. Wood et al. (73) demonstrated
that ellagic acid is a highly potent inhibitor of mutagenesis by bay-region
diol epoxides (the ultimate mutagens) of polycyclic aromatic hydrocarbons in
bacteria and mammalian cells. Chlorogenic acid, caffeic acid and ferulic acid
are also effective inhibitors but their activities are about 80-300 times
i ^
lower than that of ellagic acid. The mechanism of inhibition by these plant
phenolics appears to involve direct reaction with the reactive bay-region diol
epoxides detoxifying them in the process. Besides direct interactions, plant
phenolics may indirectly modulate the mutagenicity of nitrosation reaction
products through inhibition or catalysis of the nitrosation reaction (see Vol.
IIIA, Section 5.2.1.2.5.1.6). Tannic acid, gallic acid and chlorogenic acid,
for example, have been shown to suppress the mutagenicity of a nitrosation
mixture of nitrite and methylurea (72, 74).
Teratogenicity. There is no information available on the teratogenicity
of tannins. Chaube and Swinyard (65) found that 8 daily i.p. injections of
chlorogenic acid (5-500 mg/kg body weight) or caffeic acid (40-187.5 mg/kg) on
days 5-12 of gestation did not cause maternal or fetal lethality in Wistar
rats. No central nervous system defects were observed in the offspring. How-
ever, 30 of 289 (10.4%) chlorogenic acid-exposed 21-day-old fetuses and 12 of
274 (4.4%) caffeic acid-exposed fetuses had rib abnormalities. No such
defects were observed in 356 vehicle-control fetuses.
460
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5.3.2.6.2.3 CARCINOGENICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
Carcinogenicity of Purified Tannins and Related Compounds. The carcino-
genicity of tannic acid, a hydrolyzable tannin of gallotannin class, was first
reported by Korpassy and Mosonyi (45) in 1950 shortly after the demonstration
of the liver cirrhotic activity of the compound (75). Twenty-eight young
albino rats were given subcutaneous injections of 150-200 mg tannic acid/kg
body weight once every 5 days for up to 388 days. Of the 23 rats that sur-
vived longer than 100 days, 13 developed benign liver tumors (5 with hepatoma,
6 with cholangioma and 2 with both types of tumor). Spontaneous incidence of
these tumors was extremely Low in this strain of rat. Nine of the 13 tumor-
bearing rats also developed liver cirrhosis to various degrees whereas, in the
other four, no evidence of cirrhosis was found. Despite local necrosis at the
early stage, no local tumors were observed at the injection site. Repeated
topical application of a 5% aqueous solution of tannic acid to burn-induced
skin ulcers of rats also failed to induce any tumors in 11 rats after 400
days. It was concluded that tannic acid is a weak hepatocarcinogen in rats by
the s.c. route; no firm conclusion could be made regarding the role of cir-
rhosis in carcinogenesis.
The hepatocarcinogenicity of tannic acid was confirmed by Kirby (46)
using stock mice but not with August rats (see Table LXXVIII). In the stock
mice, liver tumors were found in seven mice (starting number of mice was not
stated) one year after receiving 12 weekly s.c. injections of 0.25 ml tannic
acid solution; no local tumors were noted. In the August rats, none of the 10
rats developed tumors 1 year after receiving 12 weekly s.c. injections of 1 ml
tannic acid solution. In the same study, plant extracts containing condensed
tannins were found to induce both local sarcomas and liver tumors in rodents
after s.c. injection, whereas those containing hydrolyzable tannins induced
461
-------
Table LXXVIII
Carcinogenicity of Purified Tannins and Related Compounds
Compound
Tannic acida
Tannin (isolated
from bracken
fern)c
Gallic acid
Catechin
Chlorogenic acid
Species
and strain
Mouse, —
Mouse, C3H/A
Rat , —
Rat, August
Mouse, Swiss
albino
Mouse , —
Mouse, —
Mouse, Swiss
albino
Principal organs
Route affected
s.c .
i .m.
s .c .
topicalb
s .c .
bladder
implantation
s.c .
s .c .
bladder
implantation
Liver
None (after
18 months)
Liver
None
None (1-year
study)
Bladder
None (1-year
study)
None (1-year
study
None ( incon-
clusive)
V
References
(46)
(76)
(45)
(45)
(46)
(64)
(46)
(46)
(64)
Mainly gallotannic acid, a hydrolyzable type of tannin.
Topical application to burn-induced skin ulcer.
cThis tannin has characteristics of condensed type of tannin.
-------
only liver tumors (see subsection on "Carcinogenicity of Tannin-Containing
Plant Extracts"). The careinogenicity of tannic acid has also been tested by
intramuscular injection in C3H/A mice (0.75 mg/kg body weight, once every two
weeks for 52 weeks); no significant carcinogenic effects were observed (76).
Two components of tannins — gallic acid and catechin — both were inactive in
stock mice 1 year after 12 weekly s.c. injections (46). This suggests that
the carcinogenicity of the tannins is probably due to intact tannins rather
than their individual components. A propyl ester of gallate has been tested
by a feeding study; no convincing evidence of carcinogenicity was obtained
(see Vol. II1B, Section 5.2.2.5 and Notes Added After Completion of Section
• • >
5.2.2.5)..
Besides tannic acid, a purified condensed tannin from bracken fern was
tested for carcinogenic activity in Swiss albino mice by the bladder implanta-
tion technique (64). The compound induced a significant increase in the
incidence of bladder carcinoma (41% vs. 16% in vehicle control). Subsequent
studies using a crude fraction of this tannin (see below) suggested that it
was active only locally and could not account for the intestinal carcino-
genicity of bracken fern. In the same study, chlorogenic acid was also tested
but found to have no significant carcinogenic effects under the test condi-
tions .
Carcinogenicity of Tannin-Containing Plant Extracts. Tannins are known
to have a wide occurrence in plants, many of which have been consumed by
humans as food, beverage, or medicine. The growing epidemiologic evidence
that consumption of tannin-rich herbal teas or medicines may be associated
with high incidence of esophageal cancer (see Section 5.3.2.6.2.5) has
prompted many investigators to study the potential carcinogenicity of various
plant extracts. Thus far, extracts of over 20 different plants have been
462
-------
tested for carcinogenic activity. The results of these studies are summarized
in Table LXXIX. Virtually all these tannin-containing plant extracts display
some carcinogenic activity when tested by s.c. route but inactive when given
by oral administration. In most cases, the carcinogenic activity of the
tannin fractions (precipitated by caffeine or quinine) is higher than that of
total aqueous extracts whereas the tannin-free fractions are either less
active or inactive suggesting that tannins are the principal carcinogenic
substances in these extracts. In some cases, however, the plant extracts may
contain known carcinogens other than tannins.
Kirby (46) was the firs,t^to test the carcinogenicity of tannin extracts
from plants. Six plants — mimosa (Acacia mollissima), myrtan (Eucalyptus
redunca, quebracho (Schinopsis lorentzii), chestnut (Castanea sativa), valonea
(Quercus aegilops), myrobalans (Terminalia chebula) — were used. The former
three are known to contain condensed tannins whereas the latter three are
known to contain hydrolyzable tannins. Both local sarcomas and liver tumors
were found in stock mice receiving s.c. injection of tannin extracts from
mimosa, myrtan and quebracho whereas only liver tumors developed in mice
receiving tannins from chestnut, valonea and myrobalans. In C57 mice or
August rats, s.c. injection of quebracho and mimosa extracts induced a low
incidence of local sarcoma whereas myrobalan and chestnut extracts were
inactive. Feeding of 0.1-0.5% solutions of quebracho and myrtan extracts to
mice for 6 months did not induce any tumors after 1 year. These results indi-
cated that all of the tannin extracts tested display some carcinogenic activ-
ity in mice after s.c. injection. Condensed tannins induced sarcomas at the
injection site as well as liver tumors whereas only liver tumors were produced
by hydrolyzable tannins. The greater local carcinogenic activity of condensed
tannins is consistent with the finding of Armstrong _et_ _al_. (63) that condensed
463
-------
page
Table LXXIX
Carcinogenic ity of Extracts of Plants Known to Contain Tannins
Type of
plant extract
Species
and strain
Route0
Care inogen ic i t yc
References
Acacia villosa (watapana shimaron), root; source: Curasao, West Indies
TAB Rat, NIH Black B.C. (2 mg) Local sarcomas (30/60) (77, 78)
Hamster, Syrian cheeck pouch Esophageal papillomas (?) (28)
oral None . (28)
>
TF Rat, NIH Black s.c. (1 mg) Local sarcomas (29/60) (78)
Acacia mollissima (mimosa)
TAB Mouse, — s.c. Liver tumors, local sarcomas (46)
Rat, August s.c. Local sarcomas (46)
Areca catechu (betel nut) , seeds; source: South India
TAB Rat, NIH Black s.c. (12 mg) Local sarcomas (30/30) (26)
Camellia sinesis (common tea or Assam tea), leaves; source: Assam, India
TF Rat, NIH Black s.c. (8 mg) Local sarcomas (21/28) (79)
Castanea sativa M. (chestnut)
TF Mouse, — s.c. Liver tumors '""* (46)
oral None (after 1 year) (46)
-------
page.
Table LXXIX (continued)
Type of Species
plant extract8 and strain Route0 Careinogenicityc References
Diospyros virginiana (persimmon) , unripe fruits or leaves; source: South Carolina
TAE (fruit) Rat, NIH Black s.c. (8 mg) None (79)
TAB (leaf) Rat, NIH Black s.c. (15 mg) Local sarcomas (17/30) (26)
TF-1 (fruit) Rat, NIH Black s.c. (8 mg) Local sarcomas (21/29) (79)
TF-2 (fruit) Rat, NIH Black s.c. (6 mg) None ' (79)
Eucalyptus redunca (myrtan)
TF Mouse, — s.c. Local sarcomas, Liver tumors (46)
oral None (46)
Hamamelis virginiana (witch hazel), leaves; source: South Carolina
TAE Rat, NIH Black s.c. (10 mg) Local sarcomas (3/30) (26)
Rat, F344 dermal None (80)
Mouse, B6C3F} dermal None (80)
Krameria ixina (cadia del perro), plant without root; source: Curacao, West Indies and Brazil
TAE Rat, NIH Black s.c. (4 mg) Local sarcomas (20/60) (77, 78, 81)
oral None (81)
-------
Table LXXIX (continued)
page
Type of
plant extract'
Species
and strain
Route0
Care inogen ic it yc
References
Krameria ixina (continued)
TF Rat, NIH Black s.c. (2 rag)
TFF Rat, NIH Black s.c. (8 rag)
Local sarcomas (28/60)
None
Krameria triandra (rhatani) , root
TAB Rat, NIH Black
TF Rat, NIH Black
Local sarcomas (30/60)
Local sarcomas (28/60)
(28)
(77, 78)
s.c. (5 mg)
s.c. (2 mg)
Limonium nashii (march rosemary), root; source: South Carolina
TF Rat, NIH Black s.c. (2 mg) Local sarcomas (13/29)
Liquidambar styracj.flua (sweet gum), leaves; source: South Carolina
TAE Rat, NIH Black s.c. (10 mg) Local sarcomas (20/28)
TF Rat, NIH Black s.c. (5 mg) Local sarcomas (26/29)
Melochia tomentosa (basora corra), root; source: Curacao, West Indies
TAE Rat, NIH Black s.c. Local sarcomas
Myrica cerifera (southern bayberry and sweet myrtle/wax myrtle), bark or leaves; source: South Carolina
TF (bark) Rat, NIH Black s.c. (2 mg) Local sarcomas (8/27) (79)
TFF (bark) Rat, NIH Black s.c. (10 mg) Local sarcomas (10/29) (79)
TAE (leaves) Rat, NIH Black s.c. (8 mg) None (26)
(78)
(78)
(79)
(79)
(79)
(77)
-------
Table LXXIX (continued)
page
Type of
plant extract
Species
and strain
Route*
Care inogen ic it yc
Pteridium aquilinum (bracken fern) ; source: Bolu, Turkey
TF
Rat, F344
8 .C .
oral
oral
Local sarcomas
None
Intestinal tumors
TFF Rat, F344
Quercus aegilops (valonea)
TF Rat, August s.c. Liver tumors
Quercus falcata pagodaefolia (cherry bark oak), bark; source: South Carolina
TAE Rat, NIH Black s.c. (8 mg) Local sarcomas (30/30)
TF Rat, NIH Black s.c. (4 mg) Local sarcomas (28/28)
Rhus cppallina (shining sumac), root; source: South Carolina
TAE Rat, NIH Black s.c. (15 mg)
Schinopais lorentzii (quebracho, sulphited)
TF Rat, August s.c.
Mouse, — s.c.
Mouse, C57 s.c.
Mouse, — oral
References
Local sarcomas (10/30)
Local sarcomas
Local sarcomas, liver tumors
Local sarcomas
None
(67)
(67)
(67)
(46)
(79)
(79)
(26)
(46)
(46)
(46)
(46)
-------
page'
Table LXXIX (continued)
Type of
plant extract8
Species
and strain Route0
Carcinogenicity0 References
Terminal ia chebula R. (my rob a Ian)
TF
Uncaria gambir
TAB
BAE
Powder
Rat, August s.c.
Mouse, — s.c.
(gambier)
Mouse, C3H/HeH s.c.
Mouse, C3H/HeN s.c.
Mouse, C3H/HeN oral
None (46)
Liver tumors (46)
>
None (cited
Local sarcoma ( c i tgd
None (cited
in 77)
in 77)
in 77)
^_
aThe abbreviations used are: TAE, total aqueous extract; TF, tannin fraction; TF-1, methanol-soluble tannin
fraction; TF-2, acetone-soluble tannin fraction; TFF, tannin-free fraction; BAE, buffered alcoholic extract.
Known to contain other carcinogens.
cStudies where dosages are given can be directly compared since the dosages (mg extract injected once per
week for lifetime or until tumor appeared) and the tumor incidences were reported in comparable studies of
Kapadia, Morton and associates are listed.
-------
tannins are held at the site of injection more firmly than hydrolyzable
tannins.
Studies by Morton, Kapadia and associates (26, 28, 77-79, 81) have
focused on plants used as herbal tea or medicine by natives of several geo-
graphic areas (Curacao, West Indies; Rio Grande do Sul, Brazil; Coro region of
Venezuela; and South Carolina, U.S.A.) with unusually high incidence of eso-
phageal cancer. Virtually all the tannin-containing plants tested — watapana
shimaron, persimmon, witch hazel, cadia del perro, rhatani, marsh rosemary,
sweet gum, basora corra, southern bayberry, cherry bark oak, shining sumac —
display carcinogenic activity after s.c. injection, inducing local sarcoma in
NIH Black rats. In most cases, the carcinogenic activity is higher in the
tannin fraction (isolated by caffeine or quinine precipitation) than the total
extract and is lower or absent in the tannin-free fraction (see Table LXXIX)
suggesting that tannins are the principal carcinogenic substance present in
these plant extracts. The extracts of watanapa shimaron (Acacia villosa) were
also tested in Syrian hamsters (28). Topical application of the extracts to
check pouch (1 g pellet held in place by steel wire) for lifespan led to
induction of an esophageal papilloma in 1 of 7 animals. Oral administration
of concentrated watanapa shimaron tea (applied to the base of the animal's
tongue) was without' carcinogenic effect. Oral administration of extracts of
cadia del perro (Krameria ixina) to NIH Black rats also failed to induce
tumors (81). The extracts of witch hazel (Hamamelis virginiana) was recently
tested under the U.S. National Toxicology Program (80). Preliminary report
indicated that Hamamelis water had no carcingoenic activity in F344 rats and
B6C3F^ mice by the dermal route.
In addition to the above plants, the tannin extract from the common tea
(Camellia sinesis) was tested by s.c. route in NIH Black rats and found to
464
-------
induce local sarcoma (79). The total aqueous extract of betel nut (Areca
catechu) was active as a local carcinogen (26); it is not known to what extent
tannins may contribute to the carcinogenicity of betel nut. It is possible
that tannins may act synergistically with other carcinogens present in the
betel nut (see Section 5.3.2.6.1.3). The tannin fraction isolated from
bracken fern (Pteridium aquilinum) also induced local sarcoma after s.c.
injection to F344 rats (67). The tannin present was isolated and found to
induce urinary bladder carcinoma after bladder implantation (see Table
LXXVIII). However, the systemic carcinogenic effects of bracken fern (see
Section 5.3.2.1.3) apparently cannot be directly attributed to the tannin.
• •*
Oral administration of the tannin fraction to F344 rats was noncarcinogenic,
but a similar treatment using a tannin-free fraction led to the induction of
intestinal tumors characteristic of bracken fern carcinogenicity.
Modification of Careinogenesis. The carcinogenicity of tannins may be
modified by dietary factors. A diet high in protein (30% casein) and low in
fat has been shown to partially protect rodents against the hepatocarcinogenic
effect of tannic acid, whereas a low protein-high fat diet has the opposite
effect (44, 82). There is also some suggestive epidemiologic evidence that
milk protein reduces the possible esophageal carcinogenic effect of tannin-
rich tea in heavy drinkers (see Section 5.3.2.6.2.5). The protective effect
of protein has been attributed to its direct interaction with tannins. Tannic
acid acts synergistically with 2-acetylaminofluorene (AAF) in the induction of
liver tumors in rodents (44, 82); over 92% (26/28) of rats given tannic acid
(s.c.) plus AAF (oral) develop liver tumors within 6 months, compared to only
28% (8/28) of rats given AAF alone (data on tannic acid alone were not
given). Korpassy (44) suggested that the synergism may be due to promotion of
AAF hepatocarcinogenesis by tannic acid-induced liver cirrhosis.
465
-------
Like simple phenolics (see Vol. IIIB, Section 5.2.2.5.3.4), tannins and
related compounds are ambivalent modifiers of carcinogenesis by a variety of
chemical agents. As discussed above, tannic acid enhances AAF hepatocarcino-
genesis. The common tea (Camellia sinesis) has been shown to promote
benzo[a]pyrene-induced skin carcinogenesis in mice (83). On the other hand,
caffeic acid, ferulic acid and/or chlorogenic acid inhibit benzol a]pyrene-
induced neoplasia in forestomach (84) and lung (85) of mice. Moreover,
ellagic acid is a potent inhibitor of benzo[a]pyrene-induced lung carcino-
genesis (85) and of 7,12-dimethylbenz[a]anthracene- or 3-methylcholanthrene-
induced skin carcinogenesis (85, 86). The mechanisms of inhibition by these
plant phenolics may involve inhibition of carcinogen activation, enhancement
of detoxification and/or blockage of interaction of the ultimate carcinogen(s)
with critical cellular macromolecules (73, 87-92). The details of these
studies will be discussed in a Section 6, Vol. IV. Tannins and related plant
phenolics can also modify the formation of N-nitroso carcinogens through
catalysis or inhibition of the nitrosation reaction; depending on the experi-
mental conditions, both types of modification have been reported (58, 72; see
also Vol. IIIA, Section 5.2.1.2.5.1.6).
5.3.2.6.2.4 METABOLISM AND MECHANISM OF ACTION
There is a scarcity of information on the metabolism of tannins. Gallic
acid derivatives, mainly 4-methoxygallate, have been identified as metabolites
of tannic acid in animals. Pyrogallol has also been detected, formed presum-
ably by the decarboxylation and dehydroxylation of gallate (93). Carob
(Ceratonia siliqua) tannins yield gallate metabolites in rat urine resulting
from the hydrolyzable tannins present, but the condensed catechin tannins por-
tion does not appear to undergo metabolism (see 42). The metabolic fate of
ellagic acid in the rat has been studied (94). After oral administration,
466
-------
ellagic acid yields 3,8-dihydroxy-6H-dibenzo[b ,d]pyran-6-one and an unidenti-
fied metabolite in the urine; however, after i.p. injection, only one uniden-
tified metabolite (a third metabolite) was detected. The formation of
3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one requires intestinal microflora (not
present in germ-free rats) and apparently involves scission of one of two
O^-pyrone rings, dehydroxylation and decarboxylation. The role of metabolism
in carcinogenesis by tannins is not clear. The demonstration of the apparent
noncarcinogenicity of gallic acid and catechin by s.c. injection (46) tends to
suggest that tannins per se, rather than their metabolites, are carcino-
genic. Oxidation of tannic acid is known to reduce the toxicity (and possibly
i %
carcinogenicity) of the compound, because the polyphenolic hydroxyl groups are
effective protein binders (through multiple hydrogen bonding), whereas the
oxidized quinoid carbonyl groups are not.
The possible mechanism(s) of the carcinogenic action of tannins are not
known. Earlier studies by Oler et al. (95) showed that tannic acid does not
cause DNA damage; however, Stich and Powrie (72) reported that, in the pre-
sence of transition metals such as Mn"*"*" and Cu"1"*" (but not Fe"*"1") , tannic acid
displays clastogenic activity, inducing chromatid breaks and exchanges. The
•
mechanism of tannic acid-induced clastogenesis and its possible contributory
role to carcinogenesis remain to be studied. Tannins are reactive and can (a)
combine with proteins (through extensive hydrogen bonding as in leather tan-
ning), (b) complex with metals, (c) inhibit respiration and oxidative phos-
phorylation by altering the mitochondrial membrane (rev. in 42, 44, 54). In
addition, tannic acid has been shown to cause rough endoplasmic reticulum
(RER) degranulation (95) and liver cirrhosis (44). These effects are all
characteristic of many carcinogens and may represent possible epigenetic
mechanisms of carcinogenesis (see 96; see also Appendix V). Possible enhance-
467
-------
ment of the formation of nitrosamine carcinogens through the catalysis of
nitrosation by plant phenolics under certain conditions has been suggested as
a possible mechanism whereby plant phenolics induce carcinogenesis (58; rev.
in 72; see also Section 5.2.1.2.5.1.6). However, it should be pointed out
that, under most conditions, plant phenolics are inhibitors, rather than
catalysts, of nitrosation. Furthermore, this mechanism cannot account for the
local carcinogenic activity of tannins after s.c. injection.
5.3.2.6.2.5 ENVIRONMENTAL SIGNIFICANCE
Tannins and related plant phenolics are present in various parts of a
large number of plants (rev., i^i 42, 53, 54, 72, 97). Hydrolyzable tannins are
relatively uncommon in human food but they do occur in the brown outer skin of
%
walnut meat and may leach from wooden barrels used to store beverages (42).
As much as 500,000 kg of tannic acid are used annually as flavoring, clarify-
ing or refining agent in wine, beer, liquor, beverages, ice cream, sweets and
baked goods (53). Condensed tannins are more widespread in human food and may
occur in beverages such as cider, cocoa, tea and red wine (some red wines may
contain as much as 1 g of tannins per liter), and in fruits and vegetables
such as spinach, persimmons and bananas (42). Some herbal teas have a high
condensed tannin content (97). The estimated average daily intake of tannins
from various human diets is somewhat higher than 400 mg (42, 98). Individuals
consuming large amounts of red wine, tannin-rich teas may have tannin intake
several times higher than the average amount. A child fed his entire milk
intake as cholocate milk and eating additional candy may consume as much as
160 mg of cocoa tannins/kg body weight (99). In addition to dietary sources,
human exposure may also occur occupationally in industries (e.g., textile,
leather tanning, printing) which utilize large amount of tannins (53). Gallic
acid has been found in wines (very high amount in red wine) and teas. Choro-
468
-------
genie acid is abundant in dry tea shoots, apples, crab apples and coffee,
while caffeic acid is present in cabbage, Brussel sprouts, radish, aubergine,
carrots, celery, lettuce and chicory (see 72). The amount of chlorogenic acid
in a cup of "instant" or ground coffee extract may be as high as 260 mg (see
58).
The potential carcinogenic risk of consumption of large amounts of
tannins has been a subject of great interest and concern. The suspicion that
tannins may be carcinogenic in humans was first raised in 1943 by Korpassy and
associates (see 44) after noting a high incidence of extensive liver necrosis
in tannin-treated burn victims. This hypothesis has been considerably
strengthened during the past decade, as epidemiologic evidence is growing that
there may be a correlation between esophageal cancer and consumption of cer-
tain plant products. Among these, probably the most significant one is the
possible association between consumption of tannin-rich teas and incidence of
esophageal cancer. The common tea (Camellia sinesis) has a tannin content
ranging from 4% (for green tea) to 33% (for black tea). The tannin fraction
of common tea is carcinogenic in rodents after s.c. injection (see Section
5.3.2.6.2.3). Morton (48) noted that esophageal carcinoma was relatively
common among the Dutch about 100 years ago when they drank tea regularly with-
out milk. The incidence of this cancer subsided considerably after the Dutch
switched from tea to coffee as their national beverage. Carcinoma of the
esophagus is rare among the tea-consuming British apparently because they put
milk in their tea; the proteins present in milk bind to tannins and reduce
their potential health hazard. Thus, both tannins and, to a lesser extent,
excessive heat are suspected to be carcinogenic factors in hot tea. However,
there are at least two case reports of esophageal cancer in patients who
indulged in strong ice tea without milk (97). In Japan, a survey by Segi
469
-------
(100) showed high mortality rates from cancer of the esophagus in three
regions (the Prefectures of Nara, Wakayama and Yamaguchi) where the inhabi-
tants are known to have the habit of regularly eating steaming-hot rice gruel
cooked with tea leaves in an iron pot. He speculated that the high incidence
of esophageal cancer in these areas may be associated with (a) the physical
condition (e.g., heat) of tea-gruel, (b) its chemical constituents (especially
tannins, more reactive at higher temperatures), and/or (c) the use of iron
pots in cooking. A 10-year follow-up study by Shimizu et al. (101) confirmed
the above finding and showed that drinking tea in addition to eating tea-gruel
can further increase the carcinogenic risk. In Iran, Mahboubi and Ghadirian
• ^
(102) surveyed the tea-drinking habits in two geographic areas with different
incidences of esophageal cancer. Inhabitants in the high incidence area drink
an average of 25 cups/day all at temperatures above 50°C while those in the
low incidence area drank 10 cups/day with less than 52% above 50°C.
Tannins have also been suspected to be carcinogenic factors in at least
five geographic areas with high esophageal cancer incidence. In Djibouti (in
East Africa), esophageal cancer is prevalent only among ethnic populations who
habitually chew Abyssinian tea (Catha edulis F.) leaves (103) containing 14%
condensed tannins (104). In Rio Grande do Sul, Brazil's area of highest
esophageal cancer incidence, the gauchos (cowboys) are reported to indulge in
mate, an aromatic beverage with leaves of Ilex paraguariensis, which contains
9-12.4% tannins (105). Surveys of esophageal cancer victims on the island of
Curacao (West Indies), in the Coro region of Venezuela, and in the Low Country
of South Carolina (U.S.A.) all suggest a possible causal relationship between
consumption of certain herbal teas (for medicinal or beverage purposes) and
cancer (47-51). Most of these herbal teas contain carcinogenic tannins (see
Table LXXIX) while some also contain other carcinogens such as safrole and
related compounds (see Section 5.3.2.4).
470
-------
Tannins are also present in betel nut, bracken fern, and wood dusts which
are implicated in the causation of human oral (section 5.3.2.6.1.5), esopha-
geal (Section 5.3.2.1,5), and nasal (106; see also Section 5.3.2.4.5.)
cancers, respectively. They may act synergistically with or modify the action
of other carcinogens present in these materials. Tannins are also used in the
leather tanning industries in which excessive cancer risks have been reported
or suspected (106).
5.3.2.6.3 Plant Flavonoids
5.3.2.6.3.1 INTRODUCTION
Flavonoids constitute one* of the largest groups of naturally occurring
products distributed universally throughout the plant kingdom. About 2,000
compounds of this class (most of them occurring in plants in the form of
glycosides) have been decribed (107). Among various flavonoid compounds,
quercetin, kaempferol and their glycosides (e.g., rutin, astragalin, tiliro-
side, etc.) are most commonly found in food plants. Although most flavonoids
are generally recognized as non-toxic and harmless, the report in 1980 of the
careinogenicity of quercetin isolated from bracken fern (108) and the evidence
obtained over the last several years on the mutagenic and genotoxic activities
of some flavonoids have raised considerable concern on the safety of food
flavonoids for human health. A symposium in 1983 highlighted the latest
investigations on flavonoids present in food plants (109). The carcinogenic-
ity of quercetin and kaempferol and the genotoxic effects of some naturally
occurring flavonoids have been the subject of several reviews (e.g., 110-112).
5.3.2.6.3.2 PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL EFFECTS
5..3.2.6.3.2.1 Physical and Chemical Properties. The fundamental skeleton of
flavonoids is a 2-pheny.'.benzo-4H-pyrane nucleus, which consists of two benzene
471
-------
rings (A and B) linked through a heterocyclic pyrane ring C (see flavonol
structure in Table LXXX). Individual compounds of the class differ by the
number and distribution of the hydroxyl groups as well as by the nature and
extent of glycosylation and/or alkylation. The flavonoid compounds that occur
most frequently in food plants are those with a hydroxyl group in the C-3
position (called flavonols). Quercetin (3,5,7,3",4'-pentahydroxyflavone) and
kaempferol (3,5,7,4'-tetrahydroxyflavone) are typical flavonols with addi-
tional hydroxylation in rings A and B. More than 70 glycosides have been
characterized for quercetin and kaempferol. The most common glycosides of
quercetin present in food plants are 3-0-rutinoside (rutin), 3-0-glucoside
• >
(isoquercitrin) and 3-0-rhamnoside (quercitrin) . Astragalin and tiliroside,
which occur in bracken fern and other plants, are the 3-0-glucoside and the
3-0-(6-p-coumaroyl) glucoside of kaempferol, respectively (110, 113).
Purified quercetin and kaempferol are yellow needles with high melting
points (314°C for quercetin and 276-278 C for kaempferol). Both compounds are
soluble in alkaline solution or hot ethanol. Kaempferol is slightly soluble
in water, whereas quercetin is practically insoluble in water (111, 112).
Both rutin and catechin form minute needles from water. They melt at about
212-216°C and are soluble in hot water. Rutin is also soluble in pyridine,
formamide and alkaline solutions. Catechin is also soluble in alcohol,
acetone and acetic acid (3).
5.3.2.6.3.2.2 Biological Effects Other Than Carcinogenicity
Toxic Effects. There is little information on the toxic effects of
flavonoids. Although quercetin, kaempferol, and many other flavonols chelate
metals and inhibit several physiologically important enzyme systems (see 109,
110) , flavonoid compounds are generally assumed to be non-toxic and harm-
472
-------
less. In fact, a number of flavonoids are used by many as dietary supplements
and therapeutic agents on account of their antioxidant properties, anti-
microbial activity and other physiological and biochemical activities
presumably beneficial to health. For instance, it is well established that
synergism exists between ascorbic acid (vitamin C) and certain flavonols such
as quercetin and rutin. This is due to the formation of a flavonoid-ascorbic
acid complex resistant to oxidation and is also due to the antioxidant
property of flavonoids. The antioxidant effect of flavonoids increases with
the degree of hydroxylation of the A and B rings. Other reported pharmaco-
logical effects of flavonoids include smooth muscle relaxation, decrease in
I" •>
capillary fragility, and anti-inflammatory and anti-histaminic and diuretic
effects (114). There is also evidence that quercetin and some other flavo-
noids exhibit cytotoxicity and are specifically inhibitory to the growth of
human tumor cells in vitro and in vivo (see 110),
The oral and s.c. LD^Q values of quercetin in the mouse are 160 and 100
mg/kg body weight, respectively. However, in the rabbit intravenous admini-
stration of quercetin at a single dose of 100 mg/kg body weight or feeding
quercetin in the diet (1%) for 410 days produces no apparent toxic effects
(cited in 111). For rutin, the LDcg values in the mouse by i.p. and i.v.
injection are 200 and 950 mg/kg body weight, respectively (115).
Mutagenic Effects. Since the initial reports in 1977 (116-118) that
quercetin and several flavonols exhibit frameshift mutagenicity in Salmonella
typhimurium, large numbers of flavonoid compounds have been screened for
mutagenic potential (69, 119-122). Among more than 70 naturally occurring and
synthetic flavonoids tested, 33 were found to display positive response in the
Ames assay; among these quercetin is the most potent mutagen, inducing rever-
tants in Salmonella strains TA98, TA1537, TAli>38, TA100 and TA1535 (110, 122-
473
-------
124). The relative mutagenicities of flavonoids in the Ames test are pre-
sented in Table LXXX.
For quercetin and other flavonols, the most sensitive tester strain of S.
typhimurium is TA98. Initial structure-activity relationship analysis (69,
120) revealed that the most important structural features for the mutagenic
action of flavonoids in S. typhimurium are: (i) presence of a free hydroxyl
group in the 3-position, (ii) a double bond in the 2,3-position, and (iii) a
keto group in the 4-position. Luteolin and apigenin, two flavonoids present
in betel leaf and other plants, for instance, having the same structures as
quercetin and kaempferol, respectively, except that they do not bear a
i ^
3-hydroxyl group, are nonmutagenic in either strain TA98 or strain TA100 of j^.
typhimurium (69, 120, 121). Catechin, which lacks the double bond in the
2,3-position, as well as the keto group, is negative in all strains of J^.
typhimurium tested (117. 120) « The hydroxyl group in position 5 of quercetin
also appears to play a role in mutagenicity since derivatives of quercetin,
which lack the hydroxyl group in position 5, are only weakly mutagenic. On
the other hand, methylation of the 7-hydroxyl group of quercetin does not
affect the mutagenic activity (121). The hydroxyl groups in the 3'- and 4'-
positions of the B ring are essential to mutagenicity without S9 mix.
Flavonoids such as quercetin and myricetin (5,7,3',4',5'-pentahydroxyl-
flavone), which have free hydroxyl groups in both the 3'- and 4'-positions of
the B ring, are direct-acting mutagens requiring no metabolic activation for
activity. The three metabolites identified in the urine of rats ingesting
quercetin, namely, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid
and 4-hydroxy-3-methoxyphenylacetic acid (homovanilic acid), are not mutagenic
in strains TA98 and TA100 of j[. typhimurium (126). Further activation of
quercetin by S9 mix is carried out by soluble cytosolic (S100) enzymes and not
474
-------
p. 1 of 5
Table LXXX
Relative Mutagenicity of Flavonoid Compounds in the Ames Test
Mutagenicity"
Compound
St ructure
Wi t ho ut
activation
With
activation
Salmonella
t yphimurium
strain
Re ference
A. Flavonols and their glycosides
OH
Quercet in
5,7,3',4'-tetra-
OH
3'-0-Methyl-
5-0-Methyl-
5,7-Di-O-methyl-
4' , 7-Di-O-raethyl-
3',4'-Di-0-raethyl-
Quercetin penta-
acetate
Quercetin-3-0-
g1uco se-rhamnose
(Rut in)
Quercetin-3-0-
glucose
(Isoquercitrin)
Rhamnetin
3,5,7,3',4'-penta-
OCOCH-,
•H-f
5,3' ,4'-tri-OH,
7-OCH-,
TA98, TA100, (69, 116-124)
TA1535, TA1537
TA1538
TA98 (69)
TA98 (69)
TA98 (69)
TA98 . o9)
TA98 ( 6 i>
TA98, TA100 U is, 121 )
TA98, TA100 169. 120, 121)
TA98, TA100 ( 121 )
TA98, TA100, (69, 120, 121)
TA1537
-------
p. 2 of 5
Table LXXX (continued)
Mutagenicity3 ..
Without
Compound Structure activation
Isorhamnetin 5 , 7 ,4-tri-OH, +
With t yphimur ium
activation strain Reference
•n- TA98, TA100 (121)
Fisetin
Kaerapferol
Kaerapfero1-3-0-
glucose
(Astragalin)
Kaempfero1-3-0-
(6-_p_-coumaroyl)-
glucose
(Tiliroside)
Kaempfer id
Galangin
3'-OCH3
7,3',4'-tri-OH
5,7 ,4'-tri-OH
5,7-di-OH, 4'-OCH3
5,7-di-OH
8-Hydroxygalangin 5,7,8-tri-OH
Myricetin
5 7 3' 4' 5 '-
-* > * iJ i^ tJ
penta-OH
Myricetin hexaacetate 3,5,7,3',4',5'-
hexa-OCOCH3
Morin 5,7,2',4'-tetra-
OH
TA98, TA100, (118-121)
TA1537
TA98, TA100 (69, 118-121)
TA1537
TA98, TA100 (121)
TA98, TA100 ((121)
TA98, TA100 (120, 121)
TA98, TA100, (122)
TA1537
TA98, TA100, (122)
TA1537
TA98 (69, 119, 120.
TA100 (69, 119, 1201
TA98, TA100 (121)
TA98, TA100, (69, 119-12U
TA1537
Robinetin
Tarmar ixet in
7,3' ,4' ,5'-tetra-
OH
5,7,3'-tri-OH,
4'-OCH3
•t- TA100,
•H-+ TA98, TA100
TA1537
(120)
(69)
-------
LXXX (continued)
p. 3 of 5
Compound
B. Flavones
•
Norwogonin
Wogonin
Isowogonin
3-Methoxynor-
wogonin
8-Hydroxyf lavone
Prime tin
Ac ace tin
Apigenin triacetate
Chrysoeriol
Pedalitin
Pedalitin tetra-
acetate
Apigenin
Luteolin
Mutagenicity3
Without With
Structure activation activation
3'
2'X^vSi4'
II ^^1
8 n I'll J<
Jf~Yl\*
s'^.^iL^JJj
1 1
0
• >
5,7,8-tri-OH - ++++b
5,7-di-OH, 8-OCH3 - +-n-b
5,8-di-OH, 7-OCH3 - ++b
5,7,8-tri-OH, - +b
3-OCH3
8-OH - +b
5,8-di-OH - +b
5,7-di-OH, 4'-OCH3 - w*
5,7,4'-tri-OCOCH3 - w+
5,7,4'-tri-OH, - w+
3 ' -OCH3
5,6,3' ,4'-tetra-OH, - w+
7-OCH3
5,6,3' ,4'-tetra- - w+
OCOCH3, 7-OCH3
5,7,4'-tri-OH
5,7,3',4'-tetra-OH
Salmonella
typhimurium
st rain
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
TA100
Re ference
(122)
(121, 122)
(122)
(122)
(122)
(122)
(121)
(121)
(121)
(121)
(121)
(121)
(121)
— - • . |.
-------
Compound
Structure
p. 4 of 5
Table LXXX (continued)
Mutagenicity
Without
With
activation activation
Salmonella
t yphimurium
strain Reference
C. Flavanones
Toxifolin
Hydrorobinet in
7,4'-Dihydroxy-
flavanone
3,5,7,3' ,V-penta-
OH
2,7,3',4',51-
penta-OH
7,4'-di-OH
TA100
TA100
TA100
(120, 121)
(121)
(121)
D. An t hocyanidins
Cyanid in
Delphinidin
3,5,7,3',4'-
penta-OH
3,5,7,3',4',5'-
hexa-OH
TA98, TA100, (120)
TA1535, TA1537
TA1538
TA98, TA100, (120)
TA1535, TA1537
TA1538
-------
p. 3 of 5
Table LXXX (continued)
Mutagenicity3
Compound
Without
Structure activation
With
activation
Salmonella
typhimur ium
st rain
Reference
E. Flavanol
Catechin
OH
TA98, TA100, (117, 120)'
TA1535, TA1537
TA1538
F. Isoflavone Derivatives
HO
lectorigenin
Genist in
6-OCH3, 7-OH
7-0-glucose
w+ TA100
w-t- TA100
(121)
(123)
^utagenicity in the Ames assay: "+" = positive; "w+" - weakly positive; "-" = negative,
Unless specified, the activation system was liver postmitochondrial fraction (S-9) plus
cofactors.
Activated by liver cytosolic fraction (S-100)
cPretreated with glycosidases and then assayed in the presence of S-9 mix.
-------
by microsomal enzymes. Kaempferol and other flavonols which do not bear a
hydroxyl group in position 3', for instance, have an absolute requirement of
metabolic activation for mutagenicity (69, 119-121). A number of investi-
gators (116, 121) noted that S9 mix hydrolyzes acetyl ester bonds but not
methyl ether bonds in flavonoid compounds, since only pentaacetyl- but not
tetramethyl- or pentamethyl-quercetin is mutagenic with S9 mix. Another
important finding is that flavonol glycosides (e.g., rutin, astragalin) are
not mutagenic, unless they are hydrolyzed to free aglycones (120, 121).
In contrast to earlier observations, certain flavonoids without the
3-hydroxyl group (the flavones) are mutagenic (121, 122). However, these
mutagenic 3-deoxyflavonoids not only have different structural requirements
but also have different bacterial strain specificity and metabolic activation
requirements from those of the flavonols. They are 8-hydroxyiated or
8-methoxylated, preferentially induce mutation in strain TA100 rather than in
strain TA98, and are activated by the soluble (S100) enzymes but not by
enzymes of the microsomal cytochrome ?45o system. The most mutagenic compound
in this group is norwogonin (5,7,8-trihydroxyflavone) . 3-Deoxyflavonoids
(without the 8-hydroxy group) are only slightly mutagenic. Also, contrary to
initial findings, weakly mutagenic activity has been reported for a few
flavanones (flavonoids without the double bond between positions 2 and 3)
(121).
The mutagenic activity of quercetin has also been observed in other test
systems, namely, the £. coli DNA repair system (119, 124), the Saccharomyces
cerevisiae strain D4 gene conversion system (119), the heritable mutagenic
assay in Drosophila melanogaster (127), the cultured V79 Chinese hamster cells
(128), Chinese hamster ovary cells (129), L5178Y mouse lymphoma (130, 131) and
the micronucleus formation test in mice in vivo (132). Similarly, kaempferol
475
-------
induces sex-linked recessive lethals in Drosophila melanogaster (127), point
mutation in V79 Chinese hamster cells after metabolic activation (128),
chromosomal aberrations in Chinese hamster cells (129), and causes increased
incidence of micronucleus in bone marrow erythrocytes of mice (132). Rutin
exhibits weak mutagenicity in a spot test in Drosophila (133) but is ineffec-
tive in the production of micronuclei in bone marrow erythrocytes of mice
(132). Catechin, cyanidin and delphinidin, which are present in betel nut,
display strong clastogenic activity in Chinese hamster ovary cells (20).
Various food products and beverages are known to contain mutagenic sub-
stances (134, 135). The mutagenicity of red wine, grape juice, raisins,
• %
onions (136), Japanese pickles (137), the Japanese spice "Sumac" (138),
certain vegetables consumed in the Netherlands (139), the methanol extract of
dill weed (140), and hydrolysates of citrus fruit juice (141) and green tea
(142) has been attributed to the presence of quercetin, kaempferol and/or
myricetin.
On the other hand, there are recent reports (143, 144) that certain plant
flavonoids such as myricetin, luteolin, robinetin and catechin possess anti-
mutagenic activity for polycyclic aromatic hydrocarbons and aromatic amines in
the Ames test.
Ter,atogenic JSffects. The teratogenic potential of plant flavonoids has
not been adequately tested. A study by Willhite (145) in the rat showed that
neither a single oral dose of 2, 20, 200 or 2,000 mg/kg quercetin administered
on day 9 of gestation nor similar oral doses of quercetin given on days 6-15
of gestation bring about significant teratogenic effects. Another study (146)
demonstrated that administration of flavonoids to pregnant hamsters following
treatment with the teratogen, /i-aminopropionitrile, significantly reduces the
476
-------
incidence of skeletal anomalies in the litters. This decrease in teratogenic
response is attributed to the ability of flavonoids to prevent the inhibition
of collagen cross-linking in the embryo by p-aminopropionitrile.
5.3.2.6.3.3 CARCINOGENICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
Flavonoids, often found in our daily diet in vegetables and fruits,
possess a number of biochemical and pharmacological effects beneficial to
human health. Few, if any, have been suspected until recently to possess
carcinogenic activity. In light of the incontrovertible evidence that certain
flavonoids are mutagenic, the Japanese Ministry of Health and Welfare mandated
in 1978 a group of scientists ,^ including I. Hirono and T. Sugimura, to inves-
tigate the carcinogenic potential of flavonoids. Interest in the carcinogenic
activity of quercetin, kaempferol, rutin and tiliroside also stems from the
search for the active principle(s) responsible for carcinogenesis by bracken
fern (see Section 5.3.2.1). The results of the carcinogenicity studies on
flavonoids are summarized in Table LXXXI.
In several feeding studies using ACI strain (151) or F344 strain rats
(149, 150), mice of ddY (152) or A strain (153) and golden hamsters (157),
Hirono, Sugimura and their associates observed no significant increase of
tumor incidence in the animals given quercetin, kaempferol or rutin in the
diet (0.1-10%) for 540-850 days. These findings are in accord with the
results of several investigators (123, 154, 158), independent from the
Japanese group, observing no carcinogenic effects with quercetin or rutin fed
to rats (Fischer and Sprague-Dawley) or mice (LACA) for prolonged periods.
Implanation of cholesterol pellets containing quercetin (64, 155), rutin (155)
or tiliroside (64) into the bladder of mice did not induce a significant
incidence of neoplasms. No tumors were observed in a study in which quercetin
477
-------
Table LXXXI
Carcinogenicity Studies of Some Plant Flavonoids
Compound3
Quercet in
Rut in
Kaempferol
Tiliroside
Catechin
Species and strain
Rat, Norwegian
Rat, F344, Sprague-
Dawley
Rat, Fischer, F344,
AC I
Mouse, ddY, A, LACA
Mouse, Swiss, N
Mouse, ICR/Ha Swiss
Hamster, golden
Rat, Sprague-Dawley ,
F344
Rat, Sprague-Dawley,
AC I
Hamster, golden
Rat, AC I
Mouse , Swiss
Mouse, C57
Principal organ
Route affected
oral
oral
oral
oral
bladder
implant .
topical
oral
oraj.
oral
oral
oral
bladder
implant .
s .c .
Urinary
bladder,
intest ine
Liver
None
None
None
None
None
Liver
None
None
None
None
Noneb
Reference
(108)
(147,
(123,
151)
148)
149-
(152-154)
(64,
(156)
(157)
(.148)
(151,
(157)
(150)
(64)
(46)
155)
158)
aSee Table LXXX for structural formulas.
Treated for only 12 weeks.
-------
(total dose of 25 mg dissolved in dimethylsulfoxide) was applied to the skin
of 50 female ICR/Ha Swiss mice three times weekly for 52 weeks (156). Whereas
sarcomas developed in C57 mice following s.c. injection of 1 ml tannin
extracts weekly for 12 weeks, no tumors were induced with catechin under
similar study conditions (46).
Despite the above reports documenting the negative response of flavonoids
in careinogenesis bioassays, quercetin is active in the Balb/c 3T3 cell (130)
and hamster embryo cell (159) transformation assays. Furthermore, Pamukcu and
coworkers (108, 147, 148) found both quercetin and rutin to be carcinogenic,
inducing intestinal, urinary bladder and/or liver neoplasms in the rat. In a
study using groups of 35-days-old male and female rats of the "Norwegian"
strain, multiple ileal intestinal adenomas and carcinomas were induced in 6 of
7 (86%) male and 14 of 18 (78%) female animals fed for 58 weeks a basic diet
supplemented with 0.1% quercetin. Also, urinary bladder carcinomas (absent in
the controls) developed in 5 of the 25 animals exposed to quercetin. Based on
the hisotpathological similarity of these neoplasms with those caused by
bracken fern, the authors (108) suggested that quercetin may be a participant
in carcinogenesis by bracken fern. In another study, lifetime feeding of
querceti^i or rutin to groups of female Sprague-Dawley and Fischer 344 rats at
0, 0.5, 1.0 or 2.0% in diet induced dose-dependent liver preneoplastic foci,
hepatomas and hepatocarcinomas, as well as bile duct adenomas and hemangio-
sarcomas in the animals (147, 148).
A 24-month feeding study on quercetin, carried out in Fischer 344 rats
under the U.S. National Toxicology Program, has been completed. At the time
of this writing, the animals are being diagnosed for gross and microscopic
lesions. Quercetin does not exhibit tumor promoting activity in rat urinary
bladder carcinogenesis initiated by N-nitroso-N-butyl— (4-hydroxybutyl)amine
478
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(160). It has been shown that the skin tumor initiating activity of some
polycyclic aromatic hydrocarbons in mice is inhibited by quercetin (156, 161,
162), but is enhanced by kaempferol and by myricetin (162).
5.3.2.6.3.4 METABOLISM AND POSSIBLE MECHANISMS OF ACTION
The mammalian metabolism of flavonoids has been reviewed by a number of
investigators (163-165). Studies in animals and humans have shown that
flavonoids are not well absorbed from the gastrointestinal tract. In the rat
approximately half of the orally administered dose of quercetin remains in the
intestines for 12 hours after dosing (166). Absorption from the digestive
tract of human volunteers aff.ef a single oral dose of 4 g quercetin was not
more than 1% of the dose (167). Extensive degradation of the unabsorbed
flavonoids or flavonoid glycosides by the intestinal microflora is known to
take place in the colon, yielding a large number of ring fission products
which include COo an^ various aromatic acids. Flavonoids with the 5,7,3",4'-
hydroxylation pattern are the most susceptible to degradation by the intes-
tinal microflora (168). Both intestinally absorbed and parenterally admini-
stered flavonoids are rapidly metabolized to glucuronide and sulfate conju-
gates. Quercetin, rutin, catechin and other 3',4'-0-dihydric flavonoids are
also metabolized in the liver by 3" and/or 4'-0-methylation, producing
conjugates of the corresponding 3'-0-methyl- and/or 4'-0-methyl ethers (169-
171). Although some unchanged compounds and the metabolites are also excreted
in the urine, biliary excretion of flavonoid metabolites is the major pathway
of disposition.
The pharmacokinetics of quercetin has been studied in humans (167).
Analysis of the data according to a two-compartment model (following an i.v.
injection of 100 mg quercetin) provided half-lives of about 9 minutes for the
479
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o<-phase and about 2.4 hours for the A-phase (a bi-exponential model of
clearance). The apparent volume of distribution was 0.34 liter/kg; binding of
quercetin to plasma proteins exceeded 98%. Of the i.v. dose, 0.65% was
excreted unchanged and 7.4% was excreted as conjugated metabolites in the
urine.
The mechanisms of the mutagenicity and carcinogenicity of flavonoids has
not yet been elucidated. Although quercetin, myricetin and other flavonoids
with hydroxyl groups in the 3'- and 4'-positions are mutagenic without
metabolic activation, they exhibit much greater mutagenicity after treatment
with rat liver microsomal preparations. Other flavonoid compounds require
' •% ~
metabolic activation for any mutagenic effects (see Section on Mutgenic
Effects) . This suggests that flavonoids produce their mutagenic and carcino-
genic action via the formation of ultimate mutagen(s) and carcinogen(s). The
aromatic acids (such as the 3-hydroxy-3-methyl, 3,4-dihydroxy-3-methyl and
4-hydroxy-3-methyl derivatives of phenylacetic acid) produced by the degrada-
tion of quercetin by intestinal microorganisms in mammals are non-mutagenic
(116, 126, 172). Based on the structural requirements for flavonoid muta-
genicity and the known chemical reactivity of hydroxyflavonols, McGregor and
Jurd (69) postulated a mechanism for the observed mutagenicity of quercetin
and other flavonols. This mechanism involves oxidation (probably by liver
microsomal enzymes or cytosol enzymes) of the 3'- and 4'-hydroxyl groups on
ring B of quercetin (Fig. 19, structure I) or its enolized form (Fig. 19,
structure II) to the quinonoid intermediates (Fig. 19, structures III, IV)
with subsequent tautomerization to a substituted quinone-methide (Fig. 19,
structure V). Since some quinone-methides are highly reactive alkylating
agents and are mutagenic (173), the substituted quinone-methide of quercetin
is suggested to be the reactive intermediate of quercetin involved in
480
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HO
0
Fig. 19. Proposed mechanism for the metabolic activation of quercetin
and other flavonols. [Adapted from J.T. MacGregor and L. Jurd: Mutat. Res.
_5i, 297 (1978).]
-------
mutagenicity and carcinogenicity. Further oxidation of this substituted
quinone-methide by gut microorganisms may lead to more reactive alkylating
intermediates (Fig. 19, Structure VI).
5.3.2.6.3.5 ENVIRONMENTAL SIGNIFICANCE
Flavonoids occur practically in all plant species. They can be found,
for example, in various leafy vegetables, fruits, seeds, nuts, roots, tubers
and bulbs, herbs and spices, tea, coffee, cocoa, and tobacco. The qualitative
and quantitative distribution of flavonoids in human food plants have been
periodically reviewed (e.g., 98, 107, 111-113). Among various flavonoid
classes, the flavonols, particularly those with hydroxylation in both the 31-
and 4'-positions, or in the 4'-position alone, occur most frequently in the
edible plants. Common food plants contain quercetin, kaempferol and their
glycosides (e.g., rutin) from trace amounts to several grams per kg fresh
weight; the highest concentrations are found generally in the free standing
leaves of vegetables or the skin and peel of fruits, tubers and roots. For
example, quercetin and kaempferol occur at levels of 273 and 150 mg/kg fresh
weight in lettuce, respectively; their respective concentrations are: 25 and
540 mg/kg fresh weight in brussels sprouts, 6 and 30 mg/kg fresh weight in
broccoli, and 33 and 14 mg/kg fresh weight in blackberries. Leaves of tea and
tobacco contain rutin and other glycosides of quercetin and kaempferol in
amounts up to 2% dry weight (113). The intake of flavonoids in the average
American diet has been estimated to be about 1 gm/day (98).
Certain flavonoids are also used in the compounding of a number of indus-
trial and medicinal preparations. Quercetin and kaempferol are components of
some food additives as well as of some natural dyes used in textile
industry. Rutin and quercetin are used in human medicine to decrease capil-
481
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lary fragility. Rut in, which is sold in health food stores as a nutritional
supplement, is effective in thrombophlebitis, bleeding gums and pulmonary
bleeding, decreasing clotting time of haemophilia and relieving allergy
symptoms. It is also used as a diuretic and as a cardiac stimulant and vaso-
constrictor (see 119). Catechin has been used for dyeing and tanning, as well
as treating acute viral hepatitis and other liver diseases (see 3, 144).
Skullcap (Scutellaria spp.), which contains the mutagenic substances, wogonin,
norwogonin and isowogonin, is also sold in health food stores as a tea and as
tablets for dietary supplement (see 122).
5.3.2.6.4 Other Carcinogenic Plant Substances
In addition to the better defined plant carcinogens discussed above, a
number of other plant substances are known to possess carcinogenic and/or co-
carcinogenic properties. N-Nitrosonornicotine and N-nitrosodiethanolamine
occur not only in tobacco smoke but also in unburnt tobacco. The carcinogenic
action of these nitroso compounds have been discussed in Section 5.2.1.2, Vol.
IIIA. Thiourea, which is isolated from seeds of certain plants of the genus
Laburnum, is carcinogenic toward the thyroid, liver and other organs of rats,
mice and rainbow trout (see Section 5.2.2.8, Vol. IIIB). One important source
of iodomethane and several haloalkanes and haloalkenes, which have been proved
or are suspected to be carcinogenic, is marine algae (see Section 5.2.2.1.5.2,
Vol. IIIB). Small amounts of carcinogenic polycyclic aromatic hydrocarbons
(PAHs) including benzo[a]pyrene, benz[a]anthracene and dibenz[a,h]anthracene
(see Vol. IIA), are present in vegetables and a variety of plant products (see
174-176). The biosynthesis of PAHs, however, is a subject of controversy.
While Graf and Diehl (177) showed that PAHs were synthesized during the
germination and growth of rye, wheat and lentils, other investigators were
unable to demonstrate biosynthesis of PAHs in plants (rev. in 178). The
482
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presence of PAHa in plants can be due to contamination from the soil and/or
atmospheric pollution.
Asiaticoside, a triterpene /J-giycoside isolated from the plant
' CO -
Asiaticosid*
CH3 G - Glucose
M - Mannow
Centella asiatica L., has been reported to produce increased incidence of
papillomas and sarcomas of the skin, when applied topically to hairless mice
as a 0.1% solution in benzene (179). Recently, some evidence of carcinogenic-
ity has been observed for benzyl acetate, the main constituent of "jasmine
absolute" which can be extracted from flowers of Jasminum officinale L. and
other species of Jasminum. A significantly increased incidence of squamous
cell papillomas or carcinomas of the forestomach was noted in male and female
B6C3Fi mice given benzyl acetate (500 and 1,000 mg/kg body weight) in corn oil
by gavage for up to 2 years. Similar treatment gave rise to acinar cell
adenomas of the pancreas in male F344/N rats (180).
Several other plant substances which have been tested or suspected for
carcinogenic activity are further discussed in this Section.
5.3.2.6.4.1 COLCHICINE AND DEMOCOLCINE (COLCEMID)
Although the poisonous action of the autumn crocus (Colehieurn autumnale
L.) was recognized some thousands of years ago, colchicine, the toxic prin-
ciple of the plant, was not isolated until 1820 by Pelletier and Caventou. At
one time, this basic, heterocyclic compound was thought to be nitrogenous and
was thus classified as an alkaloid. In 1934, Lits (181) first reported the
antimitotic effect of colchicine. Since then, this mitotic poison has been
studied and used extensively as an experimental tool in research in genetics,
483
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cell biology and biochemistry. Colchicine is also an anti-inflammatory agent
and is specific for the therapy of gout (182). Demecolcine (N-Desacetyl-N-
methylcolchicine; Colcemid), isolated also from Colchicum autumnale L., is
less toxic than colchicine and has been used in the treatment of chronic
granulocytic leukemia and lymphomas in addition to the treatment of gout
(182). However, laboratory investigations showed an enhanced tumorigenesis
when colchicine was given to mice before the initiating phase of two-stage
skin carcinogenesis (183). Treatment of Syrian hamster embryo cells in cul-
ture with demecolcine resulted in neoplastic transformation of the cells
(184). Recent studies suggest that aneuploidy induced by chemicals, such as
colchicine, and demecolcine, may play a role in carcinogenesis (184, 185).
Physical and Chemical Properties. Colchicine and demecolcine (Colcemid)
are polycyclic compounds which consist of a phenyl ring (A) and a tropone ring
(C) linked by a 3-carbon bridge thereby forming ring B. The two compounds are
_—. *»«j unou-v./ ""^V^ \
Colchicine R=-CO-CH3 II A! B /—NHR
Demecolcine R = -CH3
(Colcemid)
synthesized from tyrosine and phenylalanine; demethylation followed by
acetylation of demecolcine yields colchicine (186). Both compounds crystal-
lize as pale yellow prisms, scales or needles. Colchicine melts at 142-150°C
whereas demecolcine has a melting point of 186°C. Both compounds are soluble
in water, alcohol, chloroform, benzene and ether. The pKa value of colchicine
at 20°C is 12.35 (3).
Biologic and Toxic Activities. Colchicine is highly toxic toward
rodents; the LD50 values for mice and rats by parenteral administration range
from about 2 to 8 mg/kg (187-189). In humans, therapeutic doses and overdoses
of colchicine may cause nausea, vomiting, diarrhea, abdominal cramps, renal
484
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damage, circulatory collapse, delirium, convulsions, muscular weakness,
ascending paralysis and death due to respiratory failure. Side effects of
demecolcine include transient erythema, diarrhea, dermatitis and alopecia.
Prolonged use of these drugs may cause agranulocytosis and aplastic anemia
(182). It is known that colchicine arrests cell division at the metaphase by
virtue of its ability to bind to tubulin, a dimeric protein which aggregates
to form the microtubules of the mitotic spindle (rev. in 190). The structural
features of the colchicine molecule, responsible for the antimitotic activity
and the ability to inhibit tubulin polymerization, have been investigated. It
was found that removal of the 3-carbon bridge (which formSring B) from the
• ^
molecule did not affect the antimitotic activity of colchicine. However,
removal of the three vicinal methoxy groups (in ring A) from the molecule
abolishes activity; moreover, trimethylbenzene (corresponding to ring A) or
tnethoxytropone (corresponding to ring C) alone are also inactive. These
results, therefore, suggest that the methoxy groups in the phenyl ring (ring
A) and the two aromatic rings (ring A and ring C) together (in the molecule)
are necessary for the activity of colchicine (189, 191). Subsequent struc-
ture-activity relationship studies of a series of colchicine derivatives
revealed that compounds which have a six-membered methyl benzoate in place of
the seven-membered methoxytropone are equally effective in binding to tubulin
and inhibiting mitosis (191, 192). Several N-acyl and N-aroyl derivatives
also displayed similar activity as did colchicine (189).
Mutagenic and Teratogenic Activities. Colchicine and demecolcine are
negative in the Ames test (193), the specific locus mutation test in Syrian
hamster embryo cells (184) and the dominant lethal test in mice (194), indi-
cating that they do not induce gene mutations. However, they are known to
induce aneuploidy and polyploidy in cells in culture (195, 196) and possess
485
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clastogenic properties (194, 197). Embryotoxic effects of these mitotic
poisons have been shown in rats (198, 199) and rabbits (200, 201). Sieber et
al. (202) injected Swiss albino mice intraperitoneally with colchicine at a
single dose of 0.5 or 1.0 mg/kg body weight on day 6, 7 or 8 of gestation and
observed significant incidences of various cranial and skeletal malformations
in the fetuses. The teratogenic activity of colchicine has also been shown in
in vitro assay systems for teratogens (203, 204).
Carcinogenic ity and[Possible Mechanisms of Action. Neither colchicine
nor demecolcine may be regarded as definite carcinogens at the time of this
writing, although possible carcinogenicity mechanisms of these antimitotic
agents have been hypothesized. An early study (205) showed no tumor initiat-
ing activity of colchicine when applied to the skin of mice in a single dose
(1.0 mg) or in 15 weekly doses (3.6 mg totally) followed with croton oil
treatment. On the other hand, Berenblum and Armuth (183) reported an enhanced
tumorigenesis when 2.0 mg/kg body weight colchicine was injected s.c. into
mice 9 hours before initiation in a two-stage carcinogenesis study, with
urethan as initiator and a phorbol ester as promoter. Similar enhancement of
methylnitrosourea skin carcinogenesis by inhibiting cell proliferation with
hydroxyurea has been noted in mice (206). It was hypothesized that the
enhanced tumorigenesis may be due to the occurrence of a compensatory increase
of the rate of DNA replication some time after treatment with colchicine or
hydroxyurea, which may "fix" the carcinogen-DNA adducts before repair can take
place (206).
Recently, a role of aneuploidy in carcinogensis has been the focus of the
attention of some investigators. Tsutsui £t__al_. (184, 185) proposed that a
change in chromosome number may affect cellular regulation resulting in neo-
plastic development by: (a) altering gene balance and the amount of specific
486
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gene products, which may be critical in the homeostasis of cell division and
differentiation; (b) causing genetic instability in cells which, hence, become
prone to further chromosomal alterations; and (c) modifying nuclear organiza-
tion which is an important aspect in the control of DNA replication and
transcription. Demecolcin (Colcemid) , at doses that are non-cytotoxic and do
not cause mitotic inhibition, can induce both neoplastic transformation and
aneuploidy in Syrian hamster embryo cells. Moreover, the dose-response curves
for demecolcine-induced morphological transformation and aneuploidy are
similar. The morphologically transformed cells, when injected to newborn
hamsters, can produce anaplastic fibrosarcotnas (184). These results support
• ^
the hypothesis that the induction of aneuploidy may be important for neo-
plastic development; a number of other proven carcinogens (e.g., asbestos,
diethylstilbestrol) have also been found to induce aneuploidy (see Section
5.5.1.1 and also Appendix V).
5.3.2.6.4.2 ARISTOLOCHIC ACID
Aristolochic acid, a nitroaromatic compound, may be isolated from the
leaves and roots of Aristolochia clematitis L. and other Aristolochia species
(207, 208). Plants belonging to the genus Aristolochia (ariston = the best;
locheia = delivery, birth) were used by the ancient Egyptians and Greeks in
obstetrics and in the treatment of snake bites. Contemporary medicine has
used drugs prepared from Aristolochia plant extracts for the therapy of
arthritis, gout, rheumatism and festering wounds. In animal studies,
aristolochic acid, the pharmacologically active principle of these plants,
stimulates various defense mechanisms and shows anti-viral, anti-bacterial and
anti-fungal properties (209-211). It is not until recently that aristolochic
acid was found to be a potent carcinogen, evoking multiple site carcinomas in
rats within three months aster treatment (212, 213). The mutagenic and
487
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clastogenic activities of this compound have also been revealed in a number of
test systems (214-217). In view of the carcinogenic property of aristolochic
acid, the German Federal Health Office withdrew in 1981 the license of all
drugs containing this compound.
Physical and Chemical Properties. Chemically, aristolochic acid is a
mixture of at least 6 components; the major constitutents are aristolochic
acid I and aristolochic acid II (218). Both acids are nitrophenanthrene
HOOC
Aristolochic acid! R = -OCH3
Aristolochic acid E R = - H
HoC
derivatives with a methylenedioxy group; they differ from each other by a
methoxy group. Both compounds crystallize as shiny brown leaflets, soluble in
alcohol, chloroform, ether, acetone, acetic acid, aniline and alkalies,
slightly soluble in water and practically insoluble in benzene and carbon
disulfide (3).
Toxicity. The lethal doses of aristolochic acid in the mouse and the cat
after i.v. injections are 60 and 40 mg/kg body weight, respectively. An i.p.
dose of 1.5 mg/kg body weight is lethal to the rabbit (115). At sublethal
doses, aristolochic acid causes primarily kidney injuries in animals (219) and
in humans (220). The antifertility effect of aristolochic acid has been
established in animal studies (221, 222).
Mutagenicity. Robisch et_ _al_. (214) reported first that aristolochic acid
is mutagenic in strains TA1537 and TA100, but not in strains TA1535, TA1538
and TA98 of Salmonella typhimurium. Since the presence of S-9 mix did not
affect the mutagenic activity, aristolochic acid was classified as an
apparently "direct-acting" mutagen. These findings were confirmed by
488
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Schmeiser ^t_ _al_. (216) who found, additionally, that aristolochic acid has
only a very low level of mutagenicity in S. typhimurium strain TA100NR, a
nitroreductase deficient strain of TA100. These observations led the authors
(216) to suggest that the mutagenic effect of aristolochic acid in strain
TA100 of J^. typhimurium is due to the reduction of the nitro group by nitro
reductase to yield, presumably, the amino or hydroxylamino derivative.
When tested in Drosophila melanogaster, aristolochic acid induced muta-
tions in germ cells, sex-chromosome losses in mature spermatozoa and late
spermatids, and mitotic recombinations in somatic cells (217). The induction
of chromosomal aberrations and sister-chromatid exchange by aristolochic acid
in human erythrocytes in vitro has also been reported (215). However, the
compound did not induce unscheduled DNA synthesis in rat stomach mucosa in
vitro (223).
Carcinogenic ity and Mechanisms of Action. It was a chance observation in
a routine toxicological study which led to the discovery of aristolochic acid
being probably one of the most potent naturally occurring carcinogens of plant
origin. In these studies, aristolochic acid was administered orally through a
gastric tube to groups of 30 male and 30 female Wistar rats at doses of 0.1,
1.0 or 10.0 mg/kg/day. After 3 months of treatment, severe papillomatosis of
the forestomach with occasional signs of malignancy was noted in rats of the
1.0 and 10.0 mg/kg groups. Three to six months later without further treat-
ments, these rats developed significant incidences of carcinomas in the fore-
stomach, kidney and urinary bladder. Papillomas or squamous cell carcinomas
of the forestomach and renal carcinomas also occurred in the rats of both
sexes given 0.1 mg/kg/day aristolochic acid for 3 to 12 months (212, 213).
489
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The mechanisms of the carcinogenic action of aristolochic acid are not
known. The mechanisms of carcinogenicity of a number of nitroaromatic hydro-
carbons (see Appendix II) are known to involve reduction of the aromatic nitro
group to an amino or hydroxylamino group leading to the reactive arylamidonium
(nitrenium) ion. It is possible that aristolochic acid may exert its carcino-
genic effect via a similar mechanism. The mutagenicity data appear to lend
support to this view. As with safrole, the methylenedioxy group may also par-
ticipate in the carcinogenic action of aristolochic acid (see discussions in
Section 5.3.2.4.4).
5.3.2.6.4.3 COUMARINS AND LACTONES
A variety of naturally occurring substances of plant origin contain
lactone structure. This section focuses on two such compounds, coumarin and
L-ascorbic acid, and several of their related compounds because of the
HO OH
Coumarin Ascorbic acid
widespread usage of, and human exposure to, these compounds. Carcinogenicity
studies of other plant lactones such as parasorbic acid and bovolide (Vol.
IIIA, Section 5.2.1.1.6) and coumarin derivatives such as psoralen, bergapten
and xanthotoxin (Section 5.3.2.5) have been discussed in previous sections.
Plant lactones such as protoanemonin and ranunculin were predicted to be
potential carcinogens based on structure-activity relationship considerations
(Vol. IIIA, Section 5.2.1.1.6).
Coumarin (2H-l-benzopyran-2-one or o-hydroxycinnamic acid o-lactone) is
present in a variety of plants and essential oils which include tonka beans
(Dipteryx odorata) , sweet clover (Melilotus spp.), woodruff (Asperula
490
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odorata), balsam of Peru (Myroxylon pereirae), cassia (Cinnamoeim cassia) and
lavender (Lavandula offinalis). It is a white crystalline solid with fragrant
odor and burning taste. Coumarin has a melting point of 68-70°C and a boiling
point of 297-303°C; it is readily soluble in ethanol, ether and oils, but
sparingly soluble in water (0.25% at 25°C; 2% at 100°C). Coumarin dimerizes
upon long exposure to light and hydrolyzes to _o_-hydroxycinnamic acid under
alkaline conditions. Coumarin was/is used as a food additive (banned in
United States but still allowed in some European countries) and is still used
as a stabilizer and fragrance additive in perfumes, soaps and tobacco pro-
ducts; it is present in some alcoholic beverages (permitted levels 5-15 ppm)
i- ^
because of the use of sweet-scented herbs (such as woodruff) in flavoring wine
(3, 224). Coumarin has also been used as a chemotherapeutic agent for several
diseases (e.g., 225).
Coumarin has an oral LD5Q of 420 mg/kg in C3H/HeJ mice (226), 780 mg/kg
in DBA/2J mice (226), 680 mg/kg in rats (227) and 202 mg/kg in guinea pigs
(227). Its 3,4-dihydro derivative (dihydrocoumarin) appears to be substan-
tially less toxic, with oral LD50 values of 4,300 and 2,260 mg/kg in mice and
rats, respectively (227). There is some evidence that 5,7-dimethoxycoumarin
is phototoxic similarly to monofunctional furocoumarins (228). The subchronic
and chronic toxicity studies of coumarin have been reviewed by Cohen (224);
the liver and the kidney appear to be the most affected organs. Species
differences in the metabolism of coumarin have been noted (rev. in 224). In
man, baboon and phenobarbital-treated DBA/2J mice, 7-hydroxylation followed by
glucuronidation is the major route of metabolism, whereas in the rat coumarin
is mainly 3-hydroxylated and further degraded to o-hydroxyphenylpyruvic acid
and o-hydroxyphenylacetic acid.
491
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L-Ascorbic acid (vitamin C) is an ot,& -unsaturated "Jf-lactone with a
wide distribution in the plant and animal kingdoms. Good plant sources for
the vitamin include citrus fruits, hip berries, acerola and fresh tea leaf.
*
It is biosynthesized in all mammalian species except guinea pigs, primates and
humans (229). Purified L-ascorbic acid is a white crystalline solid with a
pleasant, sharp acidic taste. It has a melting point of 190-192°C, a density
of 1.65 and pKa values of 4.17 and 11.57. It is highly soluble in water
(about 33% at room temperature, 40% at 45°C and 80% at 100°C) , quite soluble
in propylene glycol, ethanol and glycerol but insoluble in most organic sol-
vents. L-Ascorbic acid is a relatively strong reducing agent and is exten-
i >
sively used as an antioxidant in processed foods. Aqueous solutions of
L-ascorbic acid are readily oxidized by air, particularly in the presence of
alkali, iron or copper (3). L-Ascorbic acid is reversibly oxidizable in the
body to its oxidation product, dehydroascorbic acid. Further oxidation yields
oxalate and C^. In addition, ascorbic acid-2-sulfate has been identified as
a metabolite in human urine (229). As may be expected from centuries of
extensive human usage, no serious toxicity of L-ascorbic acid has been
noted. The reported oral LD50 in rats exceeds 5 gm/kg body weight (230). The
most common untoward effect is diarrhea. Acidification of urine by high doses
of ascorbic acid may cause precipitation of cystine or oxalate stones in the
urinary tract (229). L-Ascorbic acid has been used in the treatment of a
wide variety of diseases (229). It has been claimed that megadose regimens of
L-ascorbic acid can prevent or cure viral respiratory infections and the
"common cold" (231) and may be beneficial in inhibition of in vivo formation
of nitrosamine carcinogens (232; see also Section 5.2.1.2.5, Vol. IIIA) or in
treatment of cancer (233) .
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Mutagenicity and Teratogenicity. Coumarin is either inactive or weakly
mutagenic in the Ames Salmonella test (234, 235). The compound induced a
slight, 2-fold increase in reverse mutation of strain TA100 at a high concen-
tration (in excess of 0.5-1.0 mg/plate) in the presence of large amounts of
S-9 from Aroclor-pretreated rodents. No significant increase in mutation was
observed at lower coumarin concentrations or in other tester strains. In
another study, coumarin was reported to be negative in the Ames test at con-
centrations up to 1.0 mg/plate (see 224). Coumarin is inactive in sex-linked
recessive lethal assay using Drosophila (236) and in unscheduled DNA synthesis
(UDS) assay using cultured rat epithelial cells (237). Two methoxy deriva-
r >
tives (7-methoxy-, 6,7-dimethoxy) of coumarin are nonmutagenic in the Ames
test with or without S-9 (121, 238). 6,7-Dimethoxycoumarin (esculin) is a
naturally occurring substance present in a widely used Nigerian medicinal
plant Afraegle paniculata (238). Its isomer, 5,7-dimethoxycoumarin
(limettin), however, has been shown to be photomutagenic, inducing frameshift
mutation in Escherichia coli and sister chromatid exchanges in CHO cells after
UVA photosensitization (239). Limettin is present in oil of bergamot (Citrus
aurantium L.) which also contains the photomutagenic and photocarcinogenic
comopund, 5-methoxypsoralen (see Section 5.3.2.5).
Coumarin has been shown to be devoid of teratogenic activity in mice
(240), rats (241) and rabbits (242) at 10-400 times the therapeutic dose of
coumarin-rutin combination used by humans. An increase in stillbirths and
delayed ossifications was seen in offspring of mice given 0.25% coumarin in
the diet. Coumarin gave positive results in an in vitro teratogen screening
bioassay using cultured embryonic Drosophila cells (243).
L-Ascorbic acid is not mutagenic in the standard Ames test with and with-
out metabolic activation by S-9 mix from rodent liver; however, at high con-
493
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centrations and in the presence of Cu++ ion, L-ascorbic acid is mutagenic
toward J^. typhimurium strain TA100 (193, 244-246). The mutagenic action of
the vitamin is attributed to Cu^-catalyzed generation of free radicals from
ascorbic acid leading to the formation of hydrogen peroxide which is mutagenic
(246). In other in vitro assays, L-ascorbic acid was positive in the
unscheduled DNA synthesis (UDS) assay (244, 247) and induced sister chromatic!
exchange (SCE) in Chinese hamster bone marrow cells (248-250). In contrast to
in vitro assays, L-ascorbic acid has consistently been shown to be inactive in
in vivo tests which included: (a) host-mediated assay using guinea pigs as
the host (exposed to vitamin C doses of up to 5.0 gm/kg body weight/day) and
1 •»
S. typhimurium TA100 as the test organism (246), (b) SCE assay in Chinese
hamster (250), (c) micronucleus and sperm morphology tests in mice (193), and
(d) dominant lethal test in rats (251). The reasons for the discrepancy
between in vitro and in vivo studies are not clear; at least one reason may be
the more effective detoxification of hydrogen peroxide under in vivo condi-
tions (e.g., by tissue catalase).
Carcinogenicity. The carcinogenicity of coumarin was first tested by
Dickens and Jones (252) by s.c. injection (2x2 mg/wk for 65 wk) to Wistar
rats; no carcinogenic effects were observed. Somewhat contradictory results
have been reported in several feeding studies. Hagan et al. (253) maintained
groups of 12 Osborne-Mendel rats of both sexes on diets containing 1,000,
2,500 or 5,000 ppm coumarin for 2 years. Bile duct proliferation, cholangio-
fibrosis and focal necrosis were observed in the livers of rats fed 2,500 or
5,000 ppm coumarin, but no tumors were noted. No significant hepatotoxic
effects developed in rats fed 1,000 ppm coumarin. In contrast to the above
study, Griepentrog and Bar (254, 255) reported that when albino rats received
5,000 ppm coumarin in the diet for 2 years, 11/12 male and 1/12 female rats
494
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which survived more than 18 months of treatment developed bile duct car-
cinomas. A few adenomas of the bile ducts were also observed in rats fed
1,000 or 2,500 ppm coumarin. In a recent study by Ueno and Hirono (256),
groups of 10-13 Syrian golden hamsters of both sexes were given 1,000 or 5,000
ppm coumarin in the diet for 2 years; no hepatocarcinogenic or hepatotoxic
effects were observed. In another 2-year chronic toxicity study, groups of 4-
8 baboons given daily oral doses of 2.5-67.5 mg coumarin/kg body weight did
not develop any sign of toxicity in the liver and various other organs
(257). [This study should not be considered to be indicative of lack of
carcinogenic activity because of the short duration of the study relative to
r •»
the lifespan of baboon.] Owing to the contradictory results and extensive
human exposure, the U.S. National Toxicology Program is retesting coumarin for
carcinogenic activity in B6C3F^ mice and F344 rats. The studies were still in
progress at the time of this writing.
Several derivatives of coumarin have been tested for carcinogenic activ-
ity. 3-Methylcoumarin appears not to be carcinogenic in rainbow trout; none
of the 120 trout .developed liver tumors after being exposed to 1 ppm of the
compound in the water for 3-15 months (258). 4-Methyl-7-ethoxycoumarin is
also inactive in rainbow trout (see Section 5.3.1.1.3.4). However, both
4-hydroxycoumarin and 4-methyl-7-ethoxycoumarin are carcinogenic in rats
inducing local sarcomas after s.c. injection. 6-Acetamidocoumarin is inactive
after oral administration to rats (see Vol. IIIA, Section 5.2.1.1.6). A
number of closely related furocoumarin compounds are photocarcinogenic as
discussed in Section 5.3.2.5.
Besides complete carcinogenesis studies, coumarin has been tested for
tumor-initiating activity and as a modifier of carcinogenesis. Roe and
Salaman (205) found coumarin to be devoid of tumor-initiating activity; no
495
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skin tumors developed in mice given repeated topical application of coumarin
and croton oil. Feuer et al. (259) showed that coumarin inhibits
7,12-dimethylbenz[a]anthracene- (DMBA) induced mammary carcinogenesis in
rats. The inhibitory effect was observed only if coumarin was given prior to
EMBA. This finding was confirmed by Wattenberg et al. (260) who, in addition,
showed that limettin (5,7-dimethoxycoumarin) is active but scopoletin
(7-hydroxy-6-methoxycoumarin) is marginally active and umbeliferone
(7-hydroxycoumarin) is inactive as inhibitor of DMBA carcinogenesis. Coumarin
is also an effective inhibitor of benzo[a]pyrene-induced forestomach carcino-
genesis while the other three compounds are not. Several 5-membered ring lac-
• •>
tones were also tested in that study. The totality of the results suggest
that the presence of at least one double bond in the lactone is required for
tumorigenesis inhibitory activity and that polar substituents diminish the
activity. Enzyme induction and scavenging of reactive carcinogenic inter-
mediates by nucleophilic ring-opened products of lactones and coumarins have
been suggested as possible mechanisms for tumorigenesis inhibition (261) .
Sparnins _et_ _al_. (262) showed that coumarin enhances glutathione-S-transferase
activity in mouse esophagus; a number of inducers of this enzyme are effective
inhibitors of benzo[a]pyrene-induced forestomach carcinogenesis. In contrast
to the above finding, Sinnhuber _£t__al.- (258) reported that 3-methylcoumarin
can significantly potentiate the hepatocarcinogenic effect of aflatoxin B^ in
rainbow trout. The potentiating effect was observed only after 12-15 months
of treatment.
Despite centuries of extensive human usage, L-ascorbic acid (vitamin C)
was suspected to have some carcinogenic potential following demonstration of
its genotoxicity in some test systems (see "Mutagenicity and Teratogenicity"
Section) and its possible cocarcinogenic activity (see discussion below). The
496
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U.S. National Toxicology Program (263) has recently completed a 2-year bio-
assay of the compound. Groups of 50 B6C3F^ mice and 50 F344/N rats of both
sexes were fed diets containing 25,000 or 50,000 ppm L-ascorbic acid (>97%
pure) for 103 weeks. The high dose level is the highest ever recommended for
chronic studies. The estimated intake of L-ascorbic acid in rats given 50,000
ppm of the compound in the diet is 2.6 gm/day. There was an increase in the
incidence of undifferentiated (mononuclear-cell) leukemias in low dose female
rats (34% vs. 12% in control; p < 0.002); however, the effect was deemed to be
unrelated to the administration of the vitamin because no significant increase
was observed in the high dose group. It is interesting to note that, in this
r • ^
study, high dose male mice had significantly longer survival than the control
mice. It was concluded that, under conditions of this bioassay, L-ascorbic
acid was not carcinogenic for B6C3Fi mice and F344 rat of both sexes.
Ambivalent results have been obtained from studies in which L-ascorbic
acid is used as a modifier of carcinogenesis; for certain carcinogens, the
vitamin can act as both an inhibitor as well as a potentiator or promoter.
Banic (264) reported that L-ascorbic acid acts as a cocarcinogen to 3-methyl-
cholanthrene (3-MC) in guinea pigs, reducing the latent period for the induc-
tion of fibrosarcomas and liposarcomas. Ito (cited in, 263) showed that sodium
ascorbate promotes 4-hydroxybutylbutylnitrosamine-induced preneoplastic
lesions in rat bladder epithelium. An ascorbic acid-induced increase in the
severity of urothelial lesions (including hyperplasia of the transitional epi-
thelium) was also observed in 2-acetylaminofluorene-treated mice (265). While
at low 3-MC concentration, sodium ascorbate enhances 3-MC-induced cell trans-
formation in C3H/10Tj/2 cells, at high 3-MC concentrations the vitamin acts as
an inhibitor (268). Treatment of rats with ascorbate enhances the induction
of forestomach tumor by morpholine plus nitrite, but reduces the incidences of
497
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liver and lung tumors (267). In contrast to the enhancing or ambivalent
effects described above, a number of reports indicate that vitamin C is an
effective inhibitor of carcinogenesis and may be a useful agent in the treat-
ment of cancer (rev. in 232, 233, 268-270). Some details of these studies
will be touched upon in Volume IV.
5.3.2.6.4.4 ROTENONE
For centuries, the roots of Derr is spp., Lone hoc ar pus spp., Te,phrosia
spp. and other related leguminous plants have been known to natives in various
parts of the world to contain substances that are poisonous to fish. The
active principle of these plants, rotenone, was eventually isolated in 1895.
r ^
Presently, rotenone is used extensively in many countries including the United
States, Canada, Great Britain, Sweden, Finland, Norway, Israel, Brazil and
Japan as a piscicide, to control undesirable fish species, and as an insecti-
cide to control various pests on vegetables, fruits, crops, and forage. In
1978, the annual use of rotenone in the United States was estimated to be 15
million pounds (271). Because of its instability in the environment and
selective toxicity toward cold-blooded animals, rotenone was considered to be
one of the safest pesticides. In 1973, Gosalvez and Merchan (272) reported
the induction of mammary adenomas in female albino rats given i.p. injection
of low dose of rotenone daily for 42 days. The results of this study have
aroused considerable concern regarding the possibility of rotenone being an
environmental carcinogen. Reviews on the chemistry, toxicology and carcino-
genicity of rotenone have appeared for the purpose of evaluating its potential
hazards to humans (273, 274).
Physical and Chemical Properties. The chemical structure of rotenone was
determined in 1933 by La Forge et al. (275). It is an isoflavone derivative
with a steroid-like structure belonging to the group of rotenoids. The
498
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Rotenon*
compound crystallizes in the orthorhombic form melting at 163-166 C; it is
insoluble in water, but soluble in many organic solvents. In the presence of
light and air, rotenone undergoes rapid decomposition, which increases with
the temperature (274).
Toxicity. Rotenone is a well characterized respiratory inhibitor, which
uncouples oxidative phosphorylation in mitochondria. At high concentrations,
rotenone inhibits electron transport at multiple sites in the respiratory
chain, whereas at low concentrations the inhibition is more limited and site-
i • >
specific. It is also known that retenone arrests cell division by binding to
tubulin, preventing the assembly of the microtubules. Data on the toxicity to
various animal species show that rotenone exhibits considerably higher
toxicity to insects and fishes than to mammals. While lethal doses for
various species of fish range from 8-100 iig/liter water, an oral dose of 2
g/kg does not produce any toxic effects on rabbits. The LDjQ of rotenone for
rats is 60-130 mg/kg orally and 2-5 mg/kg intraperitoneally (115, 274). It
has been estimated that a level of 10 ppm rotenone may be safe for human
consumption. Ingestion of large doses may cause gastrointestinal irritation,
nausea and vomiting. Direct contact occasionally causes dermatitis and con-
junctivitis. Inhalation of rotenone dust can cause severe respiratory
difficulties followed by convulsion, tremor and death (3, 274).
Mutagenicity. Rotenone is not mutagenic in five strains (TA98, TA100,
TA1535, TA1537 and TA1538) of Salmonella typhimurium and in one strain (WP2)
of Escherichia coli (276). It also fails to induce unscheduled DNA synthesis
in cultured human fibroblasts (277). However, Hilton and Walker (278)
reported that extensive DNA damage occurs upon exposure of mouse leukemia
cells and HeLa cells to 10 M rotenone.
499
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Teratogenicity. Being a potent mitochondrial respiratory inhibitor and a
spindle tubule poison, rotenone causes abnormalities in chick embryos in vitro
(279) and produces fetotoxicity in rats (280, 281) and hamsters (282). Oral
administration of rotenone at a dose of 5 mg/kg to Wistar rats on days 6-15 of
pregnancy produces significantly increased frequency of various skeletal aber-
rations in the fetuses (281).
Careinogenicity. Although several early studies (rev. in 273) found no
significant tumor incidence in rats or mice fed rotenone in the diet at doses
up to 100 ppm, Gosalvez and Merchan (272) reported in 1973 that mammary
adenomas developed in 24 of 35 surviving female albino rats (inbred strain)
i- ^
six to eleven months after receiving daily i.p. injections of rotenone (1.7
mg/kg body weight) for 42 days. Subsequent studies conducted by Gosalvez and
coworkers (283) using Wistar rats obtained similar results; furthermore,
mammary fibroadenotnas were induced in Wistar rats 4-11 months after they
received 0.2 mg/rat rotenone (dissolved in 0.1 ml sunflower oil) by gavage
daily for 45 days, followed by 0.3 mg rotenone daily for 15 days by the same
route. Upon histological examination, some of the rotenone-induced tumors
showed localized areas with adenocarcinotnatous transformation and the tumors
were transplantable (272, 283). It is noteworthy, however, that in these
experiments, deficient diets particularly low in riboflavin content (3.2 ppm)
were used. When Wistar rats were fed diets rich in riboflavin (13 ppm) and
other vitamins, no tumors were observed (273). The negative carcinogenic
response of Wistar and Sprague-Dawley rats and Syrian golden hamsters to
rotenone in the studies conducted by Freudental ^t__al_. (284) for the U.S.
Environmental Protection Agency was believed to be due — in addition to
inadequate dosage and time of treatment — to the use of an enriched diet
(273). Nonetheless, the U.S. Environmental Protection Agency removed rotenone
in 1981 from its "rebuttable presumption against registration" list.
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Two carcinogenesis bioassays on rotenone have been carried out in the
U.S. National Toxicology Program (285). In a study in which groups of 72
female Wistar rats were injected i.p. with rotenone at doses of 0, 2.5 or 5.0
yuMole/kg body weight, 5 days/week for 8 weeks followed by 60 weeks of observa-
tion, no dose related lesions were found in the treated animals. In another
study, groups of Fischer rats and 6603?! mice of both sexes were fed rotenone
in the diet (38 and 76 ppm for rats; 200 and 600 ppm for mice) for life. Pre-
liminary data show an increased incidence of thyroid tumors and parathyroid
tumors in male rats and a higher frequency of parathyroid neoplasms in female
rats than in the controls. No dose-related tumors were found in the treated
mice. , ^
Metabolism and Possible Mechanisms of Action. The absorption, distribu-
tion and disposition of C-labeled rotenone have been studied in the rat by
oral and i.v. administration. During the first 6 days after treatment more
than 95% of the radioactivity was excreted in the feces, and only low levels
of radioactivity was present in the liver, kidney and bone (286). Rats and
mice and in vitro liver mixed-function oxidase systems metablize rotenone to
various hydroxylated metabolites (287, 288). Many of the metabolites are
thought to be conjugated and are of reduced biological activity. On the basis
of the observations that rotenone induces an increase in noradrenaline in the
brain and elevated levels of growth hormone, estrogen and somatomedins in the
serum of rats, it was postulated that a hormonal mechanism may be involved in
the induction of tumors by rotenone (289).
5.3.2.6.4.5 CAPSAICIN
Capsaicin, N-(3-methoxy-4-hydroxybenzyl)-8-methylnon-trans-6-enamide (the
vanillylamide of ^ -8-methylnonenoic acid), is the pungent active principle
in fruits of various species of Capsicum (typical content: 0.12-0.53%),
501
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• CH-
Capsaicin
OH
conmonly known as chili pepper, hot pepper or red pepper. It is an important
ingredient of spicy foods throughout the world and has for years captured the
attention of many pharmacologists because of its wide array of biological
activities (290, 291).
Capsaicin forms colorless platelets at room temperature. It has a melt-
ing point of 61-65°C and a boiling point of 210-220°C under reduced pressure
of 0.01 mm Hg (3, 291, 292). It is practically insoluble in cold water,
sparingly soluble in hot water but freely soluble in organic solvents such as
ether, benzene, and chloroform. Its UV absorption spectrum shows two maxima
at 227 and 281 nm with molar extinction coefficients of 7,000 and 2,500,
respectively (3). Capsaicin has an intensely burning taste; it can be
detected by tasting at a concentration of about 10 ppm. The pungency of the
compound can be reduced by methylating the phenolic group or destroyed by
oxidation with potassium permanganate or dichromate (291). Capsaicin is quite
stable and is resistant to ordinary cooking conditions even in the presence of
some acids or alkali; prolonged cooking under pressure is required to achieve
breakdown of the compound (292).
The pharmacological properties of capsaicin have been thoroughly reviewed
by Virus and Gebhart (290) and Monsereenusorn ^_t__al_. (291). The compound has
strong local irritating effects causing prolonged sneezing and coughing when
inhaled and burning sensation when applied to skin, and creating sensations of
warmth, pain, and intolerable burning leading to gastrointestinal disorders
when swallowed. Systemically, capsaicin may affect (a) the cardiovascular and
respiratory systems causing transient bradycardia, hypotension and apnea, (b)
the thermoregulatory system causing initial stimulation of the hypothalamic
502
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thermoregulator and subsequent long-lasting desensitization, and (c) the
sensory system eliciting initial pain sensation and subsequent desensitization
of pain receptors. There is some evidence that the chemical analgesic effect
of capsaicin may involve depletion of neuropeptides such as substance P (291,
293). The acute toxicity of capsaicin in several animal species has been
studied by Glinsukon et al. (294). Substantial species differences have been
observed; the reported i.p. LD^Q (mg/kg/body weight) values in various animal
species are: guinea pig, 1.1; mouse, 6.5-7.65; rat, 9.5-13.2; rabbit, >50;
and hamster, >150. The toxicity of the compound is also greatly dependent on
the route of administration; the reported LDjQ (mg/kg body weight) values in
male mice by various routes art: intravenous, 0.56; intratracheal, 1.6;
intraperitoneal, 7.65; intramuscular, 7.8; subcutaneous, 9.0; intragastric,
60-75 or 190; intrarectal, >218; and dermal, >512. A commercial preparation
of pepper sauce (containing 0.2-0.75% capsaicin) has little or no toxicity in
rats when ingested (acute LD^Q = 23.6 ml/kg body weight) but can cause mild
skin irritation and severe eye damage in rabbits when directly applied or
instilled (295). Chronic feeding of capsaicin to rabbits caused hepatic and
renal necrosis (296) .
The metabolism of capsaicin and its sidechain-saturated dihydro deriva-
tive has been studied. The compound appears to be metabolized by the micro-
somal mixed-function oxidase system (297) and may bind covalently to proteins
(298, 299). An arene oxide has been postulated to be the reactive interme-
diate of capsaicin (298). However, a mutagenicity study by Nagabhushan and
Bhide (300) suggests that the unsaturated sidechain may contribute to the
mutagenic activity of capsaicin. An in vivo study by Miller et al. (298)
indicated covalent binding to hepatic proteins but no binding was observed in
the spinal cord or brain. Miller and coworkers suggested that whereas the
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covalent binding of capsaicin to hepatic proteins may initiate events asso-
ciated with the hepatotoxicity of the compound, the neurological effects of
the compound may not involve covalent binding.
Mutagenicity and Teratogenicity. The mutagenicity of capsaicin and chili
pepper oleoresin has been tested by Buchanan et_ _al_. (301) using the Ames
Salmonella test. Both materials are nonmutagenic toward four tester strains
(TA98, TA100, TA1535, TA1538) over a wide range of concentrations with or
without metabolic activation by phenobarbital-induced rat liver S-9 mix. Two
more recent studies by Toth£t__al_. (292) and Nagabhushan and Bhide (300),
however, showed that capsaicin is mutagenic after metabolic activation by
• >
Aroclor-induced rat liver S-9 mix. Aroclor-induced mouse liver S-9 mix fails
to activate capsaicin to mutagenic internediate(s). Comparison with vanillin
(which is nonmutagenic) suggests that the mutagenic activity appears to be
associated with the sidechain (300).
The potential teratogenicity of capsaicin has been studied by Kirby et
al. (302). No gross malformations were observed in offspring of rats given
injections of capsaicin at various stages of gestation. There is some
evidence that prenatal exposure at the late stage of gestation (days 16 and
17) may lead to a loss of fetal responsiveness to morphine and a decrease in
acid phosphatase in the substantia gelatinosa in spinal cord. Whether these
changes may have lasting neurological consequences in the postnatal life of
the rats is not known. A commercial preparation of pepper sauce (containing
0.2-0.75% capsaicin) was reported to have no teratogenic activity in rats
(295).
Careinogenicity. The carcinogenicity of capsaicin and chili pepper has
not been thoroughly studied. There is some evidence that capsaicin may be a
504
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weak carcinogen and that chili pepper may act as a co-carcinogen. Interest in
the study of capsaicin and chili pepper arose in 1935 when Bonne (303) noted
the high incidence of liver cancer among the Malays and Chinese in Java and
Sumatra. He pointed out that the very strongly spiced food consumed by these
populations may contain carcinogenic irritants. One of the spices used exten-
sively in Java is chili (Capsicum fruitescens L. and _£. annuum L.). In 1940,
Hieger (cited in 304) fed 30 mice a diet containing chili pepper and milk;
only one hepatoma was found in one mouse after 14 months. Hoch-Ligeti (304-
306) conducted several series of experiments to investigate the possible
carcinogenic effect of feeding chili pepper to rats along with changes in
dietary factors. In the first** series of experiments, rats were fed chili at a
level of 10% in a semisynthetic diet containing 7% casein as the sole protein
source. Seven of 30 rats developed neoplastic changes in the liver after 2
years, including 3 malignant tumors. Owing to the late appearance of the
tumors, the finding was considered inconclusive. In the second series of
experiments, the casein portion of the diet was replaced by "ardein," a
groundnut protein which is high in arginine and cystine and low in methionine,
and is known to promote liver cirrhosis. Neoplastic changes were observed in
the liver of 15 of 26 rats after only six months. In the third series of
experiments, excess vitamin B complex was given to rats on chili-ardein diet,
no liver cirrhosis and tumors were observed after nine months. Whereas no
firm conclusion regarding the carcinogenicity of chili pepper per se can be
drawn from these experiments, it appears that chili pepper may act as a co-
carcinogen under dietary conditions that favor tumor development. A similar
conclusions has been reached by Adamuyma (307) who found that chili pepper was
none arcinogenic by itself but increased the incidence of malignancies when fed
in conjunction with a known hepatocarcinogen.
505
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The firmest evidence for the weak carcinogenic activity of capsaicin has
been provided in 1984 by Toth^t__al_. (292). Groups of 8 Swiss albino mice
were fed diets containing 0.0625, 0.125, 0.25, 0.5 and 1% capsaicin (daily
intake, 2.37-30 mg) throughout their lifespan. Four of these mice (10%), one
in each of the four lower dose groups, developed adenocarcinoraas of the
duodenum. No such tumors occurred in 200 untreated control mice. The authors
(292) questioned the safety of human consumption of large amounts of chili
pepper and recommended further evaluation of carcinogenic risk.
5.3.2.6.4.6 CANNABIN01DS
The cannabinoids are naturally occurring phenolic compounds present in
i- •»
the plant, Cannabis sativa L., commonly known as marihuana* (marijuana).
Cannabis sativa, one of the oldest cultivated plants, originated from Central
Asia some 5,000 years ago and has since spread all over the temperate and
tropical zones of the globe. Although various parts of the plant are eco-
nomically used by humans (e.g., stems for fiber, seeds for oil), Cannabis
sativa has gained notoriety for the psychoactive substances (tetrahydrocan-
nabinols or THC) present in its flowering tops. Owing to the genetic plastic-
ity, environmental influence and human manipulation of the plant, as many as
several hundred variants of the plant have been identified. They are
generally classified, according to their THC content, as the "drug type" (THC
content 2-6%), the "fiber type" (THC < 0.25%) and the "intermediate" type.
Even within the same type of plants, the THC content may vary according to the
age of the plant and the environmental conditions. The medicinal potential of
*Depending on the method of preparation and the source of the plant, Cannabis
and its derivatives are also known as "hashish," "charas," "bhang," "ganja,"
and "majun."
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Cannabis was recognized some 2,000 years ago. In the 19th century, Cannabis
was briefly hailed as a miracle drug for nearly all malady but rapidly fell
into disrepute because of the extreme variability in potency of different lots
of Cannabis extracts and often irreproducible results. Today, despite some
clinical trials as potential antinauseant , anticonvulsant and analgesic,
Cannabis has remained a drug of interest mainly because of its mind and mood-
altering properties. Owing to its many potential health hazards and extensive
use as a social drug of abuse, Cannabis has captured the attention of the
scientific community as can be reflected by the large number of recent
reviews, monographs and symposia (308-317).
r • %
Over 60 different cannabinoids have been isolated from ^Cannabis plants
(318); most of these compounds are homoLogs or derivatives of three principal
types — A -tetrahydrocannabinol (A^-THC) > cannabinol (CBN), and cannabidiol
(CBD) — the structural formulas of which are depicted in Table LXXXII. There
are two nomenclature systems commonly used in numbering the cannabinoids —
the dibenzopyran system (used by Chemical Abstracts and adopted in this
section) and the monoterpene system (the preferred system in Europe). Thus,
A9-THC, the most psychoactive constituent in Cannabis, is referred to as
A -THC in the European literature. The physical and chemical properties of
cannabinoids have been described by Harvey (319) and Waller (313) and in the
Merck Index (3). Tetrahydrocannabinols and their precursor cannabidiols exist
in four different optically active stereoisomeric forms. The naturally occur-
ring A9-THC is in the (-)-A9-9,10-trans-form. It is a highly lipophilic
compound with a log Poct of about 3.8. Being not particularly stable, A9-THC
may be degraded by light, heat, acids and atmospheric oxygen, yielding canna-
binol as a major decomposition product. Cannabinol and cannabidiol have
melting points of 66-67°C and 76-77°C, respectively. They are practically
507
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Structural Formulas of Cannabinoid Conpounds Which Have Been Tested for
Carcinogenic Activity
11 CH
A9 - THC (with dibenzo-
pyran numbering system;
the sidechain is numbered
V. 2'. 3', 4', 5')
OH
5'
A1 - THC (with monoter-
pene numbering system;
the sidechain is numbered
1". 2". 3". 4", 5")
OH
OH
HoC—•
H9C
Cannabinol (CBN)
R~n~C5H11
Cannabidiol (CBD)
-------
insoluble in water but are soluble in alcohols, ethers, benzene, chloroform or
petroleum ether.
The toxicology of Cannabis and cannabinoids has been thoroughly reviewed
by Nahas (315) and Waller (313) recently. The acute toxicity of Cannabis and
its derivatives is very low by oral administration. In humans, only a few
cases of fatal acute intoxication have been reported after centuries of use.
However, the acute toxicity is enhanced by parenteral administration. The
LDcQ values for Cannabis extracts in mice by oral, subcutaneous and intra-
venous administration are 21.6, 11.0 and 0.18 g/kg body weight, respec-
tively. The acute toxicity of Cannabis appears to be related to its content
>• >
of THC, particularly A9-THC. The range of reported LD5Q values for A9-THC
in rats and mice are 482-2,000 mg/kg, 168-670 mg/kg and 29-100 mg/kg by oral,
i.p. and i.v. routes, respectively (see 315). The toxicity symptoms observed
in these two species are ataxia, hyperexcitability, depression, loss of right-
ing reflexes, and dyspnea pregressing to respiratory arrest. Postmortem
examination showed edema and congestion of lungs, and evidence of cardiac
dysfunction. The structure-activity relationships of cannabinoids have been
extensively studied; the readers are referred to the reviews of Harvey (319)
and Waller (313). In humans, A9-THC is the most psychoactive naturally
occurring cannabinoid, followed by A8-THC and A6a>10a-THC; cannabinol and
cannabidiol are inactive. With the exception of 11-hydroxy-A -THC, all
metabolites are either considerably less active than the parent compound or
inactive. Cannabinoids display a variety of symptoms of cellular toxicity.
Cannabidiol, cannabinol and A'-THC have all been shown to inhibit cellular
growth and macromolecular synthesis (rev. in 315) possibly by inhibiting the
transport of precursors across the plasma membrane (320). There is consistent
evidence that Cannabis smoke and a variety of cannabinoids are immune suppres-
508
-------
sants in experimental animals (321-325); the structural requirements for
immunosuppressant action are different from those for psychoactivity (324).
There is also some suggestive, but at present inconclusive, evidence that
similar iramunotoxic responses may also occur in humans (rev. in 315).
The metabolism of cannabinoids has been extensively studied (rev. in 319,
326). Many cannabinoids are substrates for the hepatic mixed-function oxidase
system and are actively metabolized. Metabolic attack may take place at
various sites of the molecule. Over 80 metabolites of A°-THC have been
identified. Allylic hydroxylation at the 11-position, followed by further
oxidation to a carboxy derivative, is the major metabolic pathway. Allylic
• %
hydroxylation at the 8** or 8/5 position, followed by further oxidation to the
8-oxo derivative, and hydroxylation at each of the five carbons of the pentyl
sidechain, followed by further oxidation of the terminal 5'-hydroxy to the
5'-carboxy metabolite, may also occur. Some of the hydroxy and carboxy
metabolites may be conjugated with glucuronic acid. An unusual method of
conjugation involves ester if icat ion of 11-hydroxy-A-THC with fatty acids.
Two minor metabolic pathways, involving reduction of the 9,10-double bond to
yield hexahydrocannabinol (327) and epoxidation to yield 9«C, 10"f-epoxyhexa-
hydrocannabinol (328), have been observed. The metabolism of cannabidiol and
cannabinol follows the same general pathways as those shown by A -THC. There
appears to be no firm evidence that the metabolism of cannabinoids may yield
more toxic intermediates. Virtually all the metabolites mentioned above are
pharmacologically inactive in tests used for monitoring psychoactivity and
appear to serve (with the exception of fatty acid conjugation) mainly to
facilitate excretion. For example, the epoxide of A -THC appears to be
fairly inert and is nonmutagenic in the Ames test (329).
509
-------
Mutagenicity. The mutagenicity of extracts or smoke condensates of mari-
huana and several pure cannabinoids has been assayed in a variety of test
systems. In the Ames Salmonella test, smoke condensates of marihuana have
been consistently shown to be mutagenic after metabolic activation, inducing
both frameshift and base-substitution mutations (330-332). In contrast, pure
A*-THC is inactive in in vitro assays with and without metabolic activation
(333-335) and in the host-mediated assay (333, 334), suggesting that the muta-
genicity of marihuana smoke is unrelated to A -THC. The epoxide of A -THC
is also inactive in the Ames test (329). The mutagenicity of marihuana smoke
condensate appears to be associated principally with the basic, nitrogen-
containing fraction of the coritiensate (331, 332) and is most likely attribut-
able to the pyrolytic products* of proteins and amino acids present in
marihuana. This view is supported by the finding that a dichloromethane
extract of marihuana per se (i.e., unburned) is not mutagenic (332). At least
part of the mutagenicity of marihuana smoke may be attributable to trace
amounts of mutagenic polycyclic aromatic hydrocarbons and nitrosamines which
are present in the smoke (336, 337), but absent in unburned marihuana (318).
In other in vitro assays, A -THC is negative in the unscheduled DNA repair
synthesis (UDS) assay (335) and fails to induce any significant increase in
the incidence of chromosome breaks or sister chromatid exchange in cultured
human lymphocytes (335, 338, 339). ll-Hydroxy-A9-THC, cannabinol and
cannabidiol are also negative in the UDS assay (335). However, there is some
evidence that A'-THC (but not cannabinol and cannabidiol) may increase the
incidence of segregational errors of chromosomes in cultured human lympho-
*See Appendix III for information on carcinogenicity and mutagenicity of
pyrolytic products of proteins and amino acids.
510
-------
cytes; the compound appears to affect the formation of microtubules and
spindles and may be considered as a mitotic poison (339, 340).
In contrast to the general lack of mutagenicity of pure cannabinoids in
in vitro tests, conflicting results have been reported in in vivo studies.
Mori shim a et_ _al_. (341) and Zimmerman and Raj (342) reported that A^-THC,
cannabinol or cannabidiol increased the formation of micronuclei in bone
marrow cells of mice; however, Legator ^t_ _al_. (333, 334) and Van Went (343)
were unable to find any evidence of A -THC- induced increase in micronucleus
formation. Morishima et al . (339) reviewed various conflicting cytogenetic
studies of cannabinoids and concluded that ^ -THC appears to be inactive as a
i >
clastogen, but may act as a mitotic disrupter.
Teratogenicity . The teratogenicity of marihuana extracts or of
has been extensively studied using a variety of test organisms; conflicting
results have been reported (rev. in 315, 344). With a few exceptions (e.g.,
345), most investigators (346-350) found crude marihuana extract or A^-THC
teratogenic in mice, inducing mainly cleft palate and exencephaly. There is
some evidence that perinatal exposure (late pregnancy and/or during lactation)
to ^ -THC, cannabinol or cannabidiol may lead to impairment of neuroendocrine
and reproductive functions of male mice during adulthood (351, 352). Simul-
taneous exposure of mice to modifiers of cannabinoid metabolism may influence
the teratogenicity of the compounds (348, 349). In contrast to mice, other
species of rodents, such as hamsters, rats and rabbits are less sensitive or
refractory to the teratogenic effect of marihuana extracts or cannabinoids.
Some early positive results in these species (353, 354) could not be confirmed
by subsequent studies (310, 355-360). Despite the lack of consistent terato-
genic effects, however, it should be noted that, in all species studied, dose-
related embryotoxic and fetotoxic effects were commonly observed (359-361).
511
-------
Careinogenicity. The potential carcinogenicity of cannabinoids has not
been thoroughly investigated. A number of reports provide limited evidence
that Cannabis (marihuana) smoke or smoke condensate ("tar") may be weakly car-
cinogenic; however, it is not known whether the weak carcinogenic effects
observed are related to cannabinoids. Studies using pure cannabinoids yielded
somewhat ambivalent results.
The evidence for potential carcinogenicity of marihuana smoke was first
presented by Magus and Harris (362) using a short-term predictive test,
"sebaceous gland destruction assay." Cannabis smoke condensate ("tar") con-
taining 5% A -tetrahydrocannabinol (A^-THC) was dissolved in acetone and
r ^
skin-painted to shaved CF-1 mice. Dose-related sebaceous gland destruction,
epidermal hyperplasia with acanthosis were noted; similar changes occurred
after exposure to carcinogenic polycyclic aromatic hydrocarbons. The dermal
carcinogenicity of Cannabis tar was subsequently demonstrated by Hoffmann et
al. (336) using Swiss albino mice. Thrice weekly skin painting of a 50%
Cannabis tar suspension for 74 weeks led to induction of 7 skin papillomas in
6 of 100 mice. The results were considered to be indicative of weak carcino-
genicity because the spontaneous incidence of skin tumors in these mice was
extremely low. The carcinogenicity of marihuana tar appeared to be lower than
that of tobacco tar, which induced 18 skin tumors (including 2 carcinomas) in
14 of 100 mice under similar conditions. Both marihuana and tobacco tar also
exhibited a significant tumor-promoting activity on mouse skin. Using
7,12-dimethylbenz[a]anthracene (DMBA) as the tumor initiator, the skin tumor
incidences were 26/60, 34/60 and 5/60 in mice receiving EMBA plus marihuana
tar, EMBA plus tobacco tar and EMBA alone, respectively. Besides skin cells,
lung cells may also be susceptible to the potential carcinogenic effects of
marihuana smoke. Rosenkrantz and Fleischman (363) reported that F344 rats
512
-------
exposed to marihuana smoke (equivalent to daily intake of 0.4-5 mg A^-THC/kg
body weight) for 87-360 days developed alveolitis and pneumonitis with evi-
dence of precancerous alterations (proliferative aberrations, granulomatous
inflammation) of bronchial epithelium. The authors (363) recommended a 2-year
inhalation study. Leuchtenberger et _al. (364, 365) exposed human and hamster
lung explants to the gas phase of marihuana smoke and observed anomalous
proliferation and malignant transformation of epithelial cells. Injection of
transformed cells into nude mice resulted in induction of fibrosarcoma. It is
not clear to what extent cannabinoids themselves contribute to the carcino-
genic effects of marihuana smoke, since marihuana smoke and tar are known to
contain trace amounts of varicAis carcinogenic polycyclic aromatic hydrocarbons
(e.g., benzo[a]pyrene, benzo[j]fluoranthene, dibenzopyrenes), nitrosamines
(e.g., dimethylnitrosamine, methylethylnitrosamine) and possible other car-
cinogenic pyrolysis products (336, 337). In fact, the benzo[a]pyrene content
of marihuana tar is 40-70% higher than that of tobacco tar (336, 337).
Very little information is available on the carcinogenicity of pure
cannabinoids. In two preliminary communications, Szepsenwol et al. (366, 367)
reported that BALB/c mice receiving weekly s.c. injections of 20 ug A'-THC or
cannabinol developed adrenocortical tumors (mostly benign). Injection-site
sarcomas were also observed in BALB/c and C57B16 mice given s.c. doses of
A -THC, cannabinol or cannabidiol, starting at the age of 5 days after
birth. The development of local sarcomas was influenced by sex hormones
whereas that of adrenocortical tumors was not. There was some evidence that
the cannabinoids may be active only as tumor initiators and may require the
promoting activity of sex hormones to bring about complete carcinogens. In
contrast to the above finding, Carchman, Munson ^t__al_. (368, 369) showed that
A -THC, A -THC and cannabinol (but not cannabidiol) display antineoplastic
513
-------
activity, retarding the growth of inoculated Lewis Lung tumor cells in mice,
thus prolonging the lifespan of the animals. The tumor growth inhibitory
effect of the cannabinoids may be related to their ability to inhibit nucleic
acid synthesis and to interfere with normal cell functions.
At present no epidemiclogic data are available for assessing the poten-
tial carcinogenic risk of human consumptions of Cannabis. One histopatho-
logical examination of bronchial biopsies from young, male heavy marihuana
smokers showed squamous metaplasia, a precancerous change usually observed in
much older, heavy tobacco smokers (315). The bacterial mutagenicity and
animal carcinogenicity of Cannabis smoke, together with the immunosuppressive
activity of cannabinoids strongly stress the need for epidemiologic studies on
chronic marihuana users.
5.3.2.6.4.7 VARIOUS PLANT OILS
The discovery of the tumorigenesis-promoting and carcinogenic activities
of croton oil,* the seed oil of Croton tiglium L. (Euphorbiaceae), has stimu-
lated considerable research not only on the elucidation of the molecular
mechanisms of carcinogenesis but on the identification of a new class of car-
cinogenic risk factors of plant origin. As discussed in Section 5.3.2.4,
safrole, as well as several structurally-related alkenylbenzene congeners car-
cinogenic toward several animal species, are components of various plant
oils. Additionally, a number of isoprenoid and cyclopropenoid compounds
isolated from a variety of plant oils have been found to possess weak carcino-
genic, co-carcinogenic and/or tumorigenesis promoting properties. Among these
*The tumorigenesis-promoting activity of the active principle of croton oil,
12-0-tetradecanoylphorbol-13-acetate (TPA), and other structurally-related
diterpene esters will be discussed in Volume IV.
514
-------
plant oils are those obtained from citrus, eucalyptus, turpentine, bergamot,
cinnamon, cashew nut and cotton seed. Owing to their pleasant odor, large
quantities of these plant oils or products derived from them are used in
perfumes, food flavorings, medicines or in industry as constituents of paints
and varnishes, as disinfectants and solvents. Low levels of cyclopropenoid
fatty acids may .also be found in some commercial salad oils and margarines. A
number of review articles (176, 370-372) provide valuable information on this
class of plant carcinogens and tumorigenesis-promoting agents.
Chemical and Physical Properties. Table LXXXIII presents the structural
formulas of the active components of some plant oils which have been investi-
i >
gated for carcinogenicity and/or tumorigenesis-promoting activity. Chemical-
ly, limonene, phellandrene, pinene, linalool and menthol are terpenes or
terpene derivatives formed from two isoprene (CcHg) units. They are the chief
constituents of a variety of essential oils which are colorless liquids with
characteristic scents, insoluble in water but miscible with ethanol. Their
boiling points range between 155°C and 198°C (3). Maivalic and sterculic acid
are two cyclopropenoid compounds occurring as glycerides in cotton seed oil.
They are also present in oils obtained from seeds or leaves of about 45
species in the plant order Malvales. These cyclopropenoid fatty acids give a
positive Halphen color reaction (characteristic of these compounds) and show a
strong spectral band at 1,008 cm and a weaker band at 1,870 cm in the
infrared spectrum. At room temperature, they undergo rapid oxidation, thermal
polymerization and isomerization (371). Fusel oils are mixtures of alcohols,
principally amyl alcohol, obtained as by-products of the fermentation of plant
materials.
Toxicity and Other Biological Effects. Except for peppermint oil, which
contains approximately 60% menthol, essential oils are mildly irritant to the
515
-------
CH,
Limonene
(Citrus oil)
HoC
a — Phellandrene
(Eucalyptus oil)
HO VM3
LJnalool
(Bergamot oil)
Menthol
(Peppermint oil)
Pinene
(Turpentine)
9l5H27
COOH
Anacardic acid
(Cashew nut oil)
9l5H27
Cardol
(Cashew nut oil)
Table LXXXIII
CH-
/
C-COOH
Sterculic acid: n = 7
(Cotton seed oil)
Malvalic acid: n = 6
(Cotton seed oil)
Active Principles of Some Plant Oils Which Have Been Tested for
pmi/or TumoriRenesis-promoting Activities
-------
skin and mucous membranes of the digestive, respiratory and urinary tracts.
Excess exposure may cause skin eruption, gastrointestinal irritation,
nephritis, bronchitis, delirium, ataxia, dizziness and coma. It has long been
known that because of the presence of malvalic acid and sterculic acid in
cotton seed-based poultry diets, the eggs of chicken maintained on such diets
develop a pink or red color in storage ("pink white" disorder) (rev. in
371). Other adverse effects of these two cyclopropenoid fatty acids on
various animal species include alteration of lipid metabolism, increased
cholesterol levels, aortic atherosclerosis and liver damage, retarded growth,
delayed sexual development, high prenatal and postnatal mortalities and mal-
formations of the lung, liver and kidney of newborn rats (cited in 373).
Sterculic acid has also been shown to possess mitogenic activity in rainbow
trout hepatocytes (374).
Carcinogenicity, Cocarcinogenicity and Possible Mechanisms of Action.
For many years after the pioneer work of Berenblum (375, 376), croton oil was
recognized only as a tumorigenesis-promotor. Subsequent studies showed, how-
ever, that — in addition to its tumorigenesis-promoting activity — croton
oil also exhibits properties of a complete carcinogen. Repeated treatment of
mice of several strains (e.g., Stock, C57, BRO, "101" and hr/hr) with 0.375-
0.5% croton oil in acetone either topically or subcutaneously resulted in
significant incidence of sarcomas at the exposure sites (e.g., 377-379).
Numerous hypotheses have been proposed to account for the action of croton
oil. Current evidence indicates that the phorbol esters present in croton oil
probably act by binding to cell surface receptors such as the epidermal growth
factor, protein kinase C, or other factors that are involved in the regulation
of cell multiplication and differentiation or in the induction of oncogenic
viruses (380).
516
-------
The testing of other plant oils for possible carcinogenic and co-
carcinogenic activity has been carried out by several investigators. A series
of experiments conducted by Roe and coworkers (370, 381, 382) showed that
repeated administration of various citrus oils (orange, lemon, lime or grape-
fruit), either by skin painting or by stomach tube, to stock mice and to "101"
strain mice pretreated with a subcarcinogenic dose of benzo[a]pyrene (BP),
7,12-dimethylbenz[a]anthracene (DMBA), or urethane gives rise to tumors of the
skin or the forestomach. In one study (381), epidermal hyperplasia and
neoplasms arose when a fraction of orange oil containing mainly d-limonene was
applied to the skin of mice without any pretreatment. The active carcinogenic
agent was proposed to be the- bydroperoxy derivative of d-limonene, since two
structurally related compounds, l-hydroperoxy-l-vinyl-cyclohex-3-ene and
l-hydroperoxy-cyclohex-2-ene, have also been found to be carcinogenic (372;
see also Section 5.2.1.7.3, Vol. IIIA).
Eucalyptus oil and one of its major constitutents, phellandrene, show
weak tumorigenesis-promoting activity toward mouse skin (370). They have not
been tested, however, for complete carcinogenic activity. Mackenzie and Rous
(383) reported that turpentine oil as well as 1-pinene (its principal con-
stituent) promote skin tumor development in the rabbit. Other investigators
(375, 384), however, could not reproduce the tumorigenesis-promoting activity
of turpentine oil in the mouse. Bergamot oil, which contains 60-70% alcohols
and esters, was inactive as a tumorigenesis-promoting agent when tested on the
mouse skin. Linalool, a main compound of bergamot oil, on the other hand,
elicited a weak tumorigenesis-promoting response (370). Furthermore, a potent
photocarcinogen, 5-methoxypsoralen (bergapten) is also known to be present in
bergamot oil (see Section 5.3.2.5). There is evidence that cashew nut oil,
which consists of anacardic acid (90%) and cardol (10%), is a potent tumori-
517
-------
genesis-promoting agent. Of the 15 mice ("101" strain) which survived for 20
or more weeks after receiving a single application of 150 ug EMBA and then
once weekly applications of 3-5% cashew nut oil on the skin, 12 developed a
total of 41 papillomas (370).
In a study under the U.S. National Toxicology Program, groups of 50
Fischer 344 rats and 50 B6C3Fi mice of each sex were given dl-menthol in the
diet at either 3,750 or 7,500 ppm (for rats) and either 2,000 or 4,000 ppm
(for mice) for 103 weeks. The tumor incidences were not significantly higher
than those of the corresponding control group (385).
Gibel et al. (386) administered fusel oil from potatoes to groups of 40
• •>
Wistar rats (3-month-old) either orally (0.5 ml) or subcutaneously (0.25 ml),
at the rate of three doses per week, for life. The mortality of the treated
animals was reported to be high, the average survival time being about 6
months. In the rats dosed orally, 10 papillomas of the forestomach and
esophagus developed; 5 papillomas and one carcinoma of the forestomach were
also found in rats receiving the subcutaneous doses. The first papilloma
appeared 8 weeks after treatment and the single carcinoma of the forestomach
was observed after 36 weeks. However, no mention was made in the report of
controls. The composition of the fusel oil used was described as: amyl
alcohol, 75%; isobutyl alcohol, 15%; jv-propyl alcohol, 3-4%; ethanol, 0.8%;
fatty acids including esters, 0.5%. The potent carcinogenic effects observed
in this study may be attributed to the other unidentified constituents of the
oil or to contamination by other carcinogenic substances.
Rainbow trout fed diets containing 50 ppm sterculic acid, 7.5% cotton oil
(0.35% sterculic and malvalic acids), 0.02% Sterculia foetide oil (49%
sterculic acid, 7% malvalic acids) or 0.10% Hibiscus syriacus oil (2%
518
-------
sterculic acid, 19% malvalic acid) for 12 months developed significant inci-
dences of liver tumors, indicating that sterculic acid and malvalic acid are
carcinogenic (373, 388, 390, 391). Enhancement of aflatoxin-induced hepato-
carcinogenesis by these cyclopropenoid fatty acids has also been repeatedly
demonstrated in rainbow trout. Compared to treatment with aflatoxins alone,
administration of sterculic and malvalic acids, or oils containing these
cyclopropenoid fatty acids, in combination with aflatoxins in the diet
produced higher incidences and more rapid growth rate of liver tumors in the
fish (258, 373, 387-390). Similarly, the syncarcinogenic effect of cyclo-
propenoid fatty acids with aflatoxins (392) and with diethylnitrosamine (393)
have also been observed in the> rat.
The mechanism by which cyclopropenoid fatty acids exert their syncarcino-
genic and carcinogenic effect has not been established. Since sterculic acid
stimulates DNA synthesis and the cell division of hepatocytes in rainbow trout
and in rat, it has been suggested that these effects may explain the potent
syncarcinogenicity of cyclopropenoid fatty acids co-administered with afla-
toxins in rainbow trout and rats (374). Structure-activity relationship
studies show that the cyclopropene ring is necessary for the syncarcinogenic
effects of sterculic and malvalic acids, since a structurally related
compound, epoxyoleic acid, which has an epoxy group but lacks the cyclopropene
ring, did not modify the carcinogenicity of aflatoxin Bj in rainbow trout
(388).
5.3.2.6.4.8 VARIOUS PLANT EXTRACTS CONTAINING UNIDENTIFIED CARCINOGENIC
SUBSTANCES
A number of plant extracts have been found to cause tumors in rats or
mice (see Table LXXXIV). However, the carcinogenic substances in these plant
519
-------
Table LXXXIV
Carcinogenic ity of Extracts of Plants Containing Unidentified Carcinogenic Substances
Botanical name
(common name)
Hel iotropium
a ng IPS perm urn
(Cocolode)
Cheno podium
ambrosiodes
( Sag r ado; wormseed
plant; Jerusalem
oak)
Diospyros virginana
(Persimmon)
Sassafras albidura
(Sassafras)
Glycine max L.
(Soybean)
Sol an urn tuberosum L.
(Potato)
Zingber mioga
("mioga")
Source
Curacao (West Indies)
Johns Island
(South Carolina)
Murrels Inlet
(South Carolina)
Food market ,
Washington, D.C.
Japan
Heidelberg (Germany)
Nara (Japan)
Ex t r ac t s
Boiling extracts
of dried plant
without root
Combined aqueous
and methylene
chloride extracts
of dried plant
without root
Hot water extracts
of leaf
Et Hanoi extract
of root bark3
Met Hanoi extract
of defatted seed
Undiluted sap from
the green tops
Methanol extracts
of the flowering
shoot
Principal
Species and organ
strain Route affected
Rat, NIH Black s.c. Local
sarcoma
Rat, NIH Black s.c. Local
sarcoma
f
Rat, NIH Black s.c. Local
sarcoma
Rat, NIH Black s.c. Local
sarcoma
Rat, Wistar oral Thyroid
Rat, BD IX i.p. Stomach
Rat, ACI oral Urinary0
bladder
Mouse, — implan- Urinary
tat ion bladder
Reference
(77)
(26)
(26)
(26)
(125)
(394)
(395)
(396)
aSafrole was removed by prior extraction with petroleum ether and methylene chloride.
Under iodine-deficient dietary conditions.
cln females only.
-------
extracts have not been fully characterized. Although many species in the
genus Heliotropium are known to contain carcinogenic pyrrolizidine alkaloids
(see Section 5.3.2.3), no compounds of this class have been detected in the
species _H. angios perm urn. The carcinogenicity of extracts of Cheno podium
ambrosioides and Diospyrps virginiana (see Table LXXXIV) cannot be ascribed to
tannins since they were reported to be free of tannins. Similarly, the car-
cinogenicity of extracts of Sassafras alb idurn cannot be attributed to the
hepatocarcinogen, safrole, because this compound was removed from the extracts
before the bioassay (26). Regarding the carcinogenicity of Glycine max L.
(soybean), chemical analysis revealed the presence of soya-saponine,* plum
saponine,* quercetin, and ge'nJstin, but also of thiourea, propylthiourea and
benz[a]pyrene in the tested extracts (125). Several volatile N-nitroso
compounds have been detected in the carcinogenic sap of the green tops of
Solanum tuberosum L. (potato) (394). Solanum alkaloids are also known to
occur in green spouts and green potato peels (397). Whether the reported
carcinogenic effects of soybean extracts and of potato sap are due to these
compounds remains to be investigated.
Unpublished observations also indicate that several decorative plants
common in the United States contain carcinogenic principles. These plants
are: thorns of Christ (Euphorbia milii), pencil tree (Euphorbia tirucalli),
caper spurge (Euphrobia lathyris), Candelabra cactus (Euphorbia lactea) and
coral plant (Jatropha multifida). On the other hand, no evidence for carcino-
genicity was found in bioassays with several plants used in Japan for human
food or herbal remedies (398-400). Some of these plants include: horsetail
*Saponine consists of a sapogenin, which may be a steroid or a triterpene, and
a sugar moiety which may be glucose, galactose, a pentose, or a methylpentose,
520
-------
fern (Equisetum arvense), osmund (Osmund japonica), ginkgo (Ginkgo biloba),
artemisia (Artemisia princeps), cacalia (Cacalia hastata) , dandelion
(Taraxacum piatycarpum), ostrich-fern (Matteuccia struthiopteris), aralia
(Aralia cordata) , lotus (Nelumbo nucifera), bamboo shoots (Phyllostachys
heterocycla), vicia (Vicia unijuga), galanga (Alpinia officinarum) , lathyrus
(Lathyrus palustris) and lycium (Lycium Chinese). Also, an aqueous extract of
the dried inner part of the bark of Quillaia saponaria fed to groups of male
and female Wistar rats for 2 years at levels up to 3.0% in the diet did not
exhibit any carcinogenic effects (401).
The careinogenicity of some water-soluble high polymers (e.g.,
i- ^
carrageenan, locust bean gum, guar gum, tara gum, gum arabic, agar, etc.) from
various botanical sources are discussed in Section 5.6.1.
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(London) 258, 627 (1975).
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(1974).
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Pharmacol. 10, 191 (1978).
521
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8. Shirname, L.P., Menon, M.M., Nair, J., and Bhide, S.V.: Nutr. Cancer
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384. Shubik, P.: Cancer Res. 10, 13 (1950).
385. NCI: "Bioassay of dl-Menthol for Possible Carcinogenicity." NCI
Technical Report, No. 98, National Cancer Institute, Bethesda,
Maryland, 1979.
386. Gibel, W., Wildner, G.P., and Lohs, K.: Arch. Geschwulstforsch. 32,
115 (1968) .
387. Sinnhuber, R.O., Lee, D.J., Wales, J.H., Landers, M.K. , and Keyl,
A.C.: J. Natl.,Cancer,Inst. 53, 1285 (1974).
548
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388. Lee, D.J., Wales, J.H., Ayres, J.L., and Sinnhuber, R.O.: Cancer Res.
28_, 2312 (1968).
389. Lee, D.J., Wales, J.H., and Sinnhuber, R.O.: Cancer Res. 31, 960
(1971).
390. Hendricks, J.D., Sinnhuber, R.O., Nixon, J.E., Wales, J.H., Masri,
M.S., and Hseih, D.P.H.: J^ Natl. Cancer Inst. 64, 523 (1980).
391. Sinnhuber, R.O., Hendricks, J.D., Putnam, G.B., Wales, J.H.,
Pawlowski, N.E., Nixon, J.E, and Lee, D.J.: Fed. Prop. 35, 505
(1976).
392. Lee, D.J., Wales, J.H., and Sinnhuber, R.O.: J. Natl. Cancer Inst.
43, 1037 (1969) • >
393. Nixon, J.E., Sinnhuber, R.O., Lee, D.J., Lander, M.K. , and Harr,
J.R. : J. Natl. Cancer Inst. 53, 453 (1974).
394. Ivankovic, S. : Experientia 34, 645 (1978).
395. Hirono, I., Mori, H., Kato, K., Hosaka, S., Aiso, S.: Cancer Lett.
15, 203 (1982).
396. Akiyama, M.: Tokyo Jikeikai Med. J. 93, 698 (1978).
397. Jadhav, S.J., Sharma, R.P., and Salunkhe, O.K.: CRC Grit. Rev.
Toxicol. 9_> 21 (1981).
398. Hirono, I., Shibuya, C., Shimizu, M., Fushimi, K. , Mori, H., and Miwa,
T.: Cann 63, 383 (1972).
399. Hirono, I., Hosaka, S., Uchida, E., Takanashi, H., Haga, M., Sakata,
M., Mori, H., Tanaka, T., and Hikino, H.: J. Food Safety 4_, 205
(1980).
400. Newberne, P.M.: J. Natl. Cancer Inst. 56. 551 (1976).
401. Drake, J.J.-P., Butterworth, K.R., Gaunt, I.F., Hooson, J., Evans,
J.G., and Gangolli, S.D.: Food Chem. Toxicol. 20, 15 (1982).
549
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SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.3.2.6
1. Stich, H.F. (ed.): "Carcinogens and Mutagens in the Environment,"
Vol. I, Food Products, CRC Press, Boca Raton, Florida, 1982, 310 pp.
2. Stich, H.F. (ed.): "Carcinogens and Mutagens in the Environment,"
Vol. Ill, Naturally Occurring Compounds: Epidemiology and
Distribution, CRC Press, Boca Raton, Florida, 1983, 194 pp.
3. Keller, R.F., and Tu, A.T. (eds.): "Handbook of Natural Toxins, Vol.
1, Plant and Fungal Toxins," Marcel Dekker, New York, 1983, 934pp.
4. International Agency for Research on Cancer: "Some Naturally
Occurring Substances',"* IARC Monographs on the Evaluation of
Carcinogenic Risk of Chemicals to Man, Vol. 10, Int. Agency Res.
Cancer, Lyon, 1976, 353 pp.
5. International Agency for Research on Cancer: "Some Food Additives,
Feed Additives and Naturally Occurring Substances," IARC Monographs on
the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 31, Int.
Agency Res. Cancer, Lyon, 1983, 314 pp.
6. International Agency for Research on Cancer: "Tobacco Habits Other
than Smoking; Betel-Quid and Areca-Nut Chewing; and Some Related
Nitrosamines," IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Man, Vol. 37, 1985, 291 pp.
7. Hirono, I.: Recent Advances in Research on Bracken Carcinogens and
Carcinogenic ity of Betel Nut. J. Environ. Sci. Health C3, 145-187
(1985).
8. Singleton, V.L.: Naturally Occurring Food Toxicants: Phenolic
Substances of Plant Origin Common in Foods. Adv. Food Res. 27, 149-
242 (1981).
550
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9. Harborne, J.B., and Mabry, T.J. (eds.): "The Flavonoids: Advances in
Research," Chapman and Hall, New York, 1982, 744 pp.
10. Harborne, J.B.: "Comparative Biochemistry of the Flavonoids,"
Academic, New York, 1967, 383 pp.
11. Brown, J.P. A Review of the Genetic Effects of Naturally Occurring
Flavonoids, Anthraquinones and Related Compounds. Mutat. Res. 75,
243-277 (1980).
12. Cohen, A.J.: Critical Review of the Toxicology of Coumarin with
Special Reference to Interspecies Differences in Metabolism and
Hepatotoxic Response and their Significance to Man. Food Cosmet.
. ^
Toxicol. 17, 277-279 (1979).
13. Haley, T.J.: A Review of the Literature of Rotenone. J. Environ.
Pathol. Toxicol. 1, 315-337 (1978).
14. Monsereenusorn, Y., Kongsamut, S., and Pezalla, P.O.: Capsaicin — A
Literature Survey. CRC Crit. Rev. Toxicol. 10, 321-339 (1982).
15. Nahas, G.G.: "Marihuana in Science and Medicine," Raven, New York,
1984, 312 pp.
16. Harvey, D.H. (ed.): "Marihuana "84. Proceedings of the Oxford
Symposium on Cannabis August 1984," IRL Press, Oxford, 1985, 852 pp.
17. Hoffmann, D., Brunnemann, K.D., Gori, G.B., and Wynder, E.L.: On the
Carcinogenic it y of Marijuana Smoke. In "Recent Advances in
Phytochemistry" (V.C. Runeckles, ed.), Vol. 9, Plenum, New York, 1975,
pp. 63-81.
18. Roe, F.J.C., and Field, W.E.H.: Chronic Toxicity of Essential Oils
and Certain Other Products of Natural Origin. Food Cosmet. Toxicol.
]3, 311-324 (1965).
551
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5.3.3 SUBSTANCES ELABORATED BY INSECTS AND PARASITES
While an increasing number of naturally-occurring carcinogens and muta-
gens are recognized as metabolites of microorganisms or constituents of
plants, rather little attention has been paid to the carcinogenic and muta-
genic potential of chemicals of animal origin.
Interest in several alkyl-jv-benzoquinones secreted by common flour
beetles of the family Tenebrionidae stems from the fact that the secretions of
these insects bring about contamination of grain, flour, cereal staples and
related food products consumed by humans. Studies of these insect excretions
have revealed the carcinogenic property of 1,4-benzoquinone and 1,4-naphtho-
• •>
quinone (see Section 5.2.1.7.4, Vol. IIIA). Various chromosomal aberrations
in vitro and in vivo were observed following treatment with 2,3-dimethyl-,
2,5-dimethyl-, and 2,3,5-tr imethyl-j^-benzoquinones, components of the excre-
tion (called "gonyleptidine") of an arachnid (Acanthopachylus aculeatus) from
the Opilionidae family (1, 2). As many other insects, the arachnid uses this
secretion as a defensive mechanism.
Edgar and associates (3-5) found that eight species of African and
Australian danaid butterflies, belonging to the genera Amauris, Danaus and
Euploea which feed on plants containing pyrrolizidine alkaloids, are able to
retain the alkaloids unmodified in their bodies for extended periods. The
pyrrolizidine alkaloids detected in these butterflies include the known car-
cinogens monocrotaline, lycopsamine, seneciphylline and intermedine (see
Section 5.3.2.3.1). These alkaloids are used by the males as pheromones dur-
ing courtship behavior and by both the males and females as chemical defense.
Canthardin (exo-1>2-cis-Dimethyl-3,6-epoxyhexahydrophthalic anhydride),
the active principle of the crude drug cantharides from Cantbaris vesicatoria
552
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Cantharidin
(Spanish fly, Russian fly, blistering fly, blistering beetle), from Myleabris
cichorii (telini fly) or from other insects of the families Meloidae,
Oedemeridae and Staphlinidae, has long been known to possess rubefacient and
vesicant effects on the skin and mucous membranes. The drug, therefore, was
employed as a counter-irritant and vesicant in both human and veterinary
medicine. This substance is no longer used as a drug because of its severe
toxic side effects. Severe gastroenteritis, nephritis, collapse and death
have been reported after ingest ion or absorption of the compound from the skin
and mucous membranes (6). When assayed with the "tetrazolium-reduction test"
(see 7), cantharidin gave values which were indicative of carcinogenic poten-
tial (8). Subsequent long-term studies by painting 32 male and female hair-
less mice (hr/hr strain) with 0.016% cantharidin dissolved in benzene, twice
weekly for the whole lifespan, resulted in the production of skin papillomas,
squamous carcinomas, reticuloses and/or malignant lymphomas in 60.3% of the
animals; only 7.3% of the matched controls painted with benzene developed some
small papillomas in the skin (8). Roe and Sal aman (9) observed 6 papillomas
in 4 of 17 surviving animals after painting 20 mice ("S" strain) with 0.01-
0.02% solution of cantharidin in acetone weekly for 15 weeks (total dose is
0.63 mg/animal) combined with 18 weekly croton oil (0.3-0.5% in acetone)
treatment on the skin. In the 20 mice painted with croton oil alone, only one
animal bore 3 skin tumors. Tumorigenesis-promoting activity of cantharidin
was reported in mouse skin after initiation with urethane (10), 7,12-dimethyl-
benz[a]anthracene (11) or 20-methylcholanthrene (8). These findings suggest
that cantharidin is a weak but complete carcinogen toward the skin and the
reticuloendothelial system of the mouse; it also promotes mouse skin tumori-
553
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genesis initiated by other carcinogens. Some early studies showed that
cantbar id in inhibits tumorigenesis initiated by carcinogenic tar (12) and by
benzo[a]pyrene (13). These effects are believed to be due to the potent cyto-
toxicity of cantharidin on initiated cells; the agent killed the cells which
otherwise would have transformed into tumor cells (8).
In 1926, a Nobel Prize recognizing the field of cancer research for the
first time was awarded to Johannes Fibiger, who in 1913 reported an associa-
tion between gastric cancer in rats and the ingestion of nematodes found in a
strain of cockroach. Subsequently, attempts to repeat Fibiger1s findings
failed and the idea of a causative association between the parasite and cancer
• ^
induction was essentially discarded. Nonetheless, it has long been suspected
that certain parasites, particularly the trematodes (flatworms), play a role
in the onset of carcinogenesis in infested animals and individuals. The high
prevalence of bladder cancer, liver cancer and other neoplasms in some areas
where Schist qsoma hematob_ium, S. mansoni, S. japonicum, S. intercalating,
Opistorchis viverrini or Clpnorchis sinensis is endemic, and the significant
pathological findings from experimental studies tend to confirm the associa-
tion between schistosomiasis and cancer (rev. in 14, 15), although the
mechanism whereby schistosomiasis plays a role in the etiology of these
neoplasms is not understood. Among the various hypotheses, one suggests the
involvement of schistosomal toxins; however, no experimental support for this
hypothesis has emerged. There are also speculations that endogenous or
exogenous carcinogenic metabolites may be produced as a result of altered
metabolism of the host tissues due to schistosomiasis. Experimental data have
demonstrated the presence, in the liver, serum or urine of schistosome-
infested animals, of elevated levels of enzymes that may activate procar-
cinogens or promutagens into their reactive intermediates in selected host
organs (16-18).
554
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In the light of our present ignorance of the chemical and toxicological
properties of most of the toxins elaborated by creatures existing in the human
environment, it seems that more attention should be directed toward this
research area in the future if all possible risks to human health are to be
identified and assessed.
REFERENCES TO SECTION 5.3.3
1. Drets, M.E., Folle, G.A., and Aznarez, A.: Mutat. Res. 102, 159
(1982).
2. Drets, M.E.: Env iron'. Xutagen. _5_, 923 (1983).
3. Edgar, J.A., and Culvenor, C.C.J.: Nature 248, 614 (1974).
4. Edgar, J.A., Cockrum, P.A., and Frahn, J.L.: Experientia 32, 1535
(1976).
5. Edgar, J.A., Boppre, M., and Schneider, D.: Experienta 35, 1447
(1979).
6. Windholz, M. (ed.): "The Merck Index," 10th ed., Merck and Co.,
Rahway, N.J., 1983.
7. Westwood, F.R.: Br. J. Cancer 37, 949 (1978).
8. Laerum, O.D., and Iversen, O.H.: Cancer Res. 32, 1463 (1972).
9. Roe, F.J.C., and Salaman, M.H.: Br. J. Cancer 9. 177 (1955).
10. Pounds, A.W.,a nd Withers, H.R.: Br. J. Cancer 17, 460 (1963).
11. Hennings, H., and Boutwell, R.K.: Cancer Res. 30, 312 (1970).
12. Berenblum, I.: J. Pathoi. Bacteriol. 40, 549 (1935).
13. Mottram, J.C.: J. Pathol. Bacteriol. 56, 391 (1944).
14. Cheever, A.W.: J. Natl. Cancer Inst. 61, 13 (1978).
15. Gentile, J.M.: Environ. Mutagen. 7, 775 (1985).
555
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16. Gentile, J.M., and DeRuiter, E.: Toxicol. Lett. 8. 273 (1981).
17. Flavell, D.J., and Lucas, S.B.: Br. J. Cancer 4, 985 (1982).
18. Gentile, J.M., Brown, S., Aardema, M., Clark, D., and Blankespoor,
H.: Arch. Environ. Health 40, 5 (1985).
556
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