CURRENT AWARENESS DOCUMENT
IHDANONE DERIVATIVES AND RELATED COMPOUNDS:
BRACKEN FERN TOXINS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by:
David Y. Lai, Ph.D.
Yin-Tak Woo, Ph.D., D.A.B.T.
Science Applications Internation Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
SAIC Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc.
Extradi vi sional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5.3.2.1 Bracken Fern Toxins
5.3.2.1.1 Introduction
Bracken fern (Pteridium aquilinum), a primitive plant belonging to the
Family Polypodiaceae (Order Pteridophyta), occurs worldwide and is cultivated
in Japan, New Zealand, Canada and in the northeastern United States for human
consumption as a vegetable or in salads. Yet, the plant contains toxins and
has long been known to cause characteristic illness in grazing animals. It
first came to the attention of oncologists when an association was noted
between chronic ingest ion of the plant and the emergence of urinary bladder
and intestinal tumors in cattle and sheep. Careinogenesis bioassays using
laboratory animals demonstrated that bracken fern not only induces tumors of
the urinary bladder and the intestine, but also neoplasms of other sites in
several species. Recent epidemiological studies have shown that human con-
sumption of bracken fern is correlated with an increased incidence of
esophageal carcinomas in Japan.
Much time and effort have been expended in the identification and charac-
terization of bracken toxins by various investigators around the world. How-
ever, the exact chemical identity of the components responsible for the toxic
and carcinogenic properties of bracken fern remains elusive. No single chemi-
cal isolated so far can mimic all aspects of the toxicity and carcinogenicity
displayed by intact bracken fern, when administered to susceptible animal
species. The toxins identified in bracken fern, suspected and/or tested for
carcinogenicity, include shikimic acid (1), pterolactam (2), pterosins and
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pterosides (3-5), ptaquiioside (aquilide A) (6-8), glycosides of quercetin
(rut in and isoquercitrin) and of kaempferol (astragal in and tilroside) (9, 10)
and tannin (11). The structural formulas of some of these toxins are shown in
Table XXXIII.
For many years, the chemical, biological and carcinogenic properties of
bracken fern have been the subjects of extensive research by several investi-
gators, particularly I.A. Evans in the United Kingdom, I. Hirono in Japan and
A.M. Pamukcu in the United States. Many of their important findings are
covered in several reviews (e.g., 12-14).
5.3.2.1.2 Physicochemical Properties and Biological Effects
5.3.2.1.2.1 PHYSICAL AND CHEMICAL PROPERTIES
There appears to be a consensus that the chemical constituents in bracken
fern which are carcinogenic are also the agents which confer to the plant its
toxicity. The compounds are heat stable and methanol-, ethanol-, chloroform-
and water-soluble. Some physicochemical properties of shikimic acid, ptero-
lactam, pterosin B, pteroside B and ptaquiioside (aquilide A) are presented in
Table XXXIV.
Shikimic acid (3,4,5-trihydroxy-l-cyclohexene-l-carboxylic acid) is a
water-soluble, white solid. It is not stable in solution and may undergo
spontaneous aromatization and oxidation. Shikimic acid, found widespread in
plants, is an intermediate in the biosynthesis of many aromatic plant consti-
tuents from carbohydrates. The chemistry, biochemistry and distribution of
this naturally occurring substance have been extensively discussed in a review
in 1965 (15).
Pterolactam (5-methoxy-2-pyrrolidone) is a novel compound isolated from
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COOH
HO
OH
Shikimic acid
H
Pterolactam
Pterosin B
(R = CH2OH)
Pteroside B
= _CH20 glucose)
HO
OH
Ptaquiloside
(Aquilide A)
Table XXXIII
Some Bracken Fern Toxins Which Have Been Tested for Carcinogenic Activity
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Table XXXIV
Physicochemical Properties of Some Bracken Fern Toxins'
Empirical
Compound formula m.p. (°C) Specific rotation
\ KBr
IR 0
" max
(cm'1)
Shikimic acid C7H1Q05 191-192
Pterolactam C5Hg02N 56-67
Pterosin B C14H18°2 109-110
Pteroside B C20H28°7 120-122
]^8 - -185° (MeOH)
I2,5 = +2.0° (CHC13)
I2,5 = -31.9° (MeOH)
]25 = -48.8° (MeOH)
Ptaquiloside C20H30Og 173-174° [OC]2)2 « -188.0° (MeOH)
(Aquil ide A) '' "*
1,700
3,300, 1,705,
1,670
3,360, 1,683,
1,605
3,400, 1,724,
1,640
aData summarized from Stavric, B., and Stoltz, D.R. [Food Cosmet. Toxicol. 14,
141-145 (1976)]; Takatori, K., Nakano, S., Nagata, S., Okumura, K., Hirono,
I., and Shiraizu, M. [Chem. Pharm. Bull. 20, 1087 (1972)]; Fukuoka, M.,
Kuroyanagi, M., Yoshihira, K., and Natori, S. [Chem. Pharm. Bull. 26. 2365-
2385 (1978)]; and Niwa, H., Ojika, M., Wakamatsu, K., Yamada, K., Hirono, I.,
and Matsushita, K. [Tetrahedron Lett. 24, 4117-4120 (1983)].
bSee Table XXXIII for structural formulas.
cPtaquiloside tetraacetate.
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bracken fern by Takatori et al. (2) in 1972. It was extracted by methanol
from young leaves of the plant. It forms colorless leaflets when recrystal-
lized from petroleum ether.
Pterosin B and pteroside B are among more than thirty 1-indanone deriva-
tives isolated from the leaves and rhizomes of bracken fern (16). The charac-
terization and absorption spectra of these compounds have been extensively
described (3-5). Ptaquiloside (aquilide A), another characteristic consti-
tuent of bracken fern, is a norsesquiterpene glucoside of the illudane type.
The compound is rather unstable and readily converts to pterosins under acidic
conditions. The half-life of ptaquiloside in 0.01 M sulfuric acid-met Hanoi at
22°C is about 2 hours. Under alkaline conditions, the glucose moiety and the
proton at position 9 are removed, forming an aglycon (designated aquiline A)
with an unsaturated bond between C-4 and C-9 (6, 8):
OH
Ptaquiloside Aikaiine ^
/"A -f-j A\ conditions _cHo
(Aquilide A) ^ 3
Aquiline A
Ptaquiloside is a possible biosynthetic precursor of pterosins and ptero-
sides. Its concentration in freeze-dried bracken fern is about 2.5 g/kg (8)
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Quercetin and kaempferol are flavonoids occurring in conjugated or uncon-
jugated forms in many plants. Tannin is a generic term referring to a group
of phenolic compounds widely distributed in the plant kindgom as heterogenous
polymeric substances. In bracken fern, the concentrations of quercetin,
kaempferol (as liberated aglycones) and tannin, in terms of g/kg dry weight,
are 0.57-0.86, 1.10-2.55 and 2.45, respectively (14). These compounds are
further discussed in Section 5.3.2.6.2 and Section 5.3.2.6.3.
5.3.2.1.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY
Toxic effects. Poisoning of cattle as a result of excessive feeding on
bracken fern was first scientifically recorded in 1893 (17, 18). Since then,
i <»
the toxic action of bracken fern on various animal species has been repeatedly
established in the field as well as demonstrated in experimental studies.
When ingested by horses, pigs, sheep, rats and pigeons, bracken fern produces
the typical nervous lesions of avitaminosis B,. The affected animals show
anorexia, staggering and incoordination. This syndrome is caused by the
presence of thiaminase in the plant and can be reversed by the administration
of thiamine.
Ingestion of bracken fern by cattle and several other animal species
results in "cattle bracken poisoning," characterized by bone marrow aplasia,
damage to the gastrointestinal mucosa and generalized hemorrhage (19). Con-
tinued feeding of the plant leads to "chronic enzootic bovine hematuria,"
resulting from characteristic lesions produced in the urinary bladder mucosa
(20). These manifestations of bracken fern toxins in cattle do not differ
significantly from those caused by ionizing radiation or radiomimetic chemi-
cals and they-cannot be prevented or remedied by supplementation with thiamine
(12, 21).
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It is possible that bracken fern contains more than one radiomimetic
principles. The acute bracken fern toxicity in cattle can be reproduced with
ptaquiloside (22) but not with bracken fern extracts containing pterosins and
pterosides (23). These findings led Hirono et al. (22) to suggest that the
causative factor in "cattle bracken poisoning" is ptaquiloside.
The shikimic acid present in bracken fern possibly accounts for the
plant's acute toxicity in cattle. The identity of shikimic acid in bracken
fern has been established by Evans and Osman (1). The compound is present at
a much higher concentration in the rhizomes than in the frond (24-26).
Shikimic acid is mutagenic, carcinogenic and lethal to mice on i.p. injection
(27), but there is little or no information on the cytologically established
radiomimetic effects of the compound. The LD5Q of shikimic acid in mice by
i.p. injection is 1 g/kg (1).
Mutagenic effects. Aqueous and organic solvent extracts of bracken fern
contain substances mutagenic to the Salmonella typhimurium strains TA100 and
TA98 after microsomal activation (7, 28). Milk from cows fed bracken fern and
urine from rats ingesting bracken fern reversed _S_. typhimurium TA100 and TA98
mutants, without the addition of S-9 mix (29, 30). Bracken fern extracts also
exhibit positive rautagenic responses in the Drosophila test (27, 31) and in
the mouse dominant lethal assay (32). They induce sister-chromatid exchange
in V79 Chinese hamster cells (7) and cause damage to DNA in gastrointestinal
epithelial cells in vitro (cited in ref. 33). The mutagenicity is substan-
tially reduced in processed bracken fern (used as a human food) after treat-
ment with salt, sodium bicarbonate or wood ash (28).
Besides flavonoids and their glycosides (discussed in Section 5.3.2.6.3),
other mutagens are believed to be present in the extracts and fractions of
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bracken fern (28, 30). Evans and Osman (1) suggested that shikimic acid may
be the mutagenic principal in bracken fern extracts, on the basis of the find-
ing that shikimic acid induces dominant lethal mutations in mice. However,
neither shikimic acid nor its bacterial and mammalian metabolites (see Fig. 9
for structural formulas) showed any activity in the Ames test with or without
metabolic activation (34-36). Furthermore, shikimic acid did not produce
chromosomal aberrations in Chinese hamster cells in culture (37) and its domi-
nant lethal effects on mice were not reproducible (38). More than twenty
pterosins, pterosides and their derivatives isolated from fronds of bracken
fern have been examined for mutagenicity using S. typhimurium and mammalian
cells (FM3A cells). None of the compounds exhibited mutagenicity in these
systems or caused chromosomal aberrations in Chinese hamster cells (16). Van
der Hoeven jet_ _al_. (8) have recently isolated from bracken fern a potent muta-
genic compound, designated aquilide A (also called ptaquiloside), which is
responsible for more than 50% of the mutagenic activity observed in a methanol
extract of the plant. Aquilide A is strongly mutagenic to J^. t yphimurium
strains TA100 and TA98 under alkaline conditions and is a potent inducer of
sister-chromatid exchange and HGPRT-deficient mutants in V79 Chinese hamster
cells, and of unscheduled DNA synthesis in human fibroblast (8).
Reproductive and teratogenic effects. Studies with mice, quail and
Drosophila have indicated that bracken fern has a sterilizing effect on male
animals (21, 27). Pregnant mice fed a diet containing 33% bracken fern showed
maternal weight loss, intrauterine growth suppression (39) and an increase in
abortion rate (40). Significant abnormalities in the ribs and the sternebrae
with retarded ossification were found in the fetuses (40). In another experi-
ment, however, when given to mice at 0.25 or 1.00 g/kg/day through day 18 of
pregnancy, shikimic acid exhibited no teratogenic effects in the newborn (41).
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5.3.2.1.3 Carcinogenicity and Structure-Activity Relationships
5.2.3.1.3.1 OVERVIEW
In advanced cases of "chronic enzootic bovine hematuria," the development
of neoplastic lesions of various forms and sizes in the urinary bladder is a
common finding. In 1965, Rosenberger and Heeschen (42) first described
changes of a polypous-tumorous nature in the bladder mucosa of five cows with
hematuria caused by bracken fern. Subsequent studies by other investigators
t
substantiated the carcinogenicity of bracken fern in the urinary bladder of
cows and, in addition, demonstrated that bracken fern contain toxins which are
potent carcinogens toward many sites in various animal species. Dried and
i %
fresh plant, or solvent extract of bracken fern, induce bladder cancer in
cows, guinea pigs, rats and mice, intestinal tumors in sheep, hamsters, guinea
pigs, Japanese quails, toads, rats and mice, lymphatic leukemia and lung
tumors in mice, mammary gland tumors in rats and hepatomas in toads (Table
XXXV). Solvent extracts of urine or milk from cattle fed bracken fern also
induced tumors of the bladder in calves, dogs, rats and mice (29, 67, 68).
Comparative studies have shown that the latent period for the induction
of intestinal tumors in rats is shorter with the immature young fern than with
the mature fern (50). All parts of the plant are oncogenic; however, the car-
cinogenicity of the rhizomes is greater than that of the fronds, which in turn
has a greater carcinogenic activity than the stalks (60). Weak but definite
carcinogenic activity still remains in processed bracken fern treated with
boiling water containing sodium bicarbonate, sodium chloride or wood ash (59).
There is some evidence that shikimic acid (1), tannin (11), quercetin and
kaempferol (69) isolated from bracken fern display carcinogenic activity (see
also Section 5.3.2.6.2 and Section 5.3.2.6.3). However other substance(s) ,
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Table XXXV
Carcinogenic ity of Bracken Fern by Oral Administration3
Species and strain
Principal organs affected
Reference
Cow, Turkish
Sheep (Swaledale x Scottish
Blackface)
Hamsters, Syrian golden
Guinea pig,
Quail, Japanese
(Cot urnix cot urnix japonica)
Toad, Egyptian
(Bufo regularis)
Mouse, Swiss
Mouse, C57BL/6
Mouse, dd
Mouse, ICR
Mouse, Swiss
Rat, Glaxo
Rat, Albino
Rat, AC I
Rat, Wistar
Rat, Fischer
Rat, Sprague-Dawley
Rat, Sprague-Dawley (CD)
Urinary bladder
Intestine
Intestine
Intestine, urinary bladder
Intestine
Intestine, liver
Lung, liver, intestine,
haematopoietic tissues
Intestine
Lung
Urinary bladder
Urinary bladder0
Intest ine
Intestine, urinary bladder
Intestine, urinary bladder
Intestine
Intestine, urinary bladder
Intestine, urinary bladder,
mammary gland
Mammary gland
(43-45)
(21)
(13, 21)
(21, 46, 47)
(21, 46)
(48)
(21, 32, 49)
(50)
(50)
(51)
(52)
(53)
(45, 54-57)
(58-63)
(16, 64)
(16, 65)
(65)
(66)
aDried or fresh bracken fern, or solvent extracts of bracken fern, were mixed
in the diet of tested animals, unless otherwise indicated.
Animals were fed powdered bracken fern mixed with a basic diet after the
implantation of a glass bead into the urinary bladder.
cPellets composed of bracken fern extract and cholesterol were surgically
implanted into the urinary bladder of the animals.
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more potently carcinogenic than these compounds, is/are believed to be present
in the plant. Pterolactam, pterosin B, pteroside B and extracts of bracken
fern containing other 1-indanone derivatives, were found to be not carcino-
genic in rats and mice (16, 50, 70). In 1984, Hirono and coworkers (71, 71)
isolated from bracken fern a novel norsequiterpene glucoside of the illudane
type, called ptaquiloside (aquilide A) (see Table XXXIII for structure), and
claimed that ptaquiloside may be the substance responsible for the hemato-
toxic, mutagenic and carcinogenic effects of bracken fern. The carcinogenic-
ity studies of some bracken fern toxins are summarized in Table XXXVI.
5.3.2.1.3.2 CARCINOGEN1CITY OF BRACKEN FERN
f %
Demonstration that ingested bracken fern is the etiological agent of
bovine bladder cancer in several regions of the world has been provided by
experimental testings in cows as well as in other animals. In a study in
which 18 cows ranging from 1.5 to 4 years of age and weighing from 100 to 150
/
kg were fed dried (300-600 gm) or fresh (400-1,000 gm) bracken fern daily, 10
surviving animals which received bracken fern at the lower dietary levels,
developed urinary bladder carcinomas, papillomas and hemangiomas after a mean
feeding period of 550 days (43); these animals also had hematuria. Similar
findings were obtained from prolonged, low-level feeding of bracken fern to
calves; the induced neoplasms were histologically indistinguishable from the
naturally occurring bovine bladder tumors (44, 45).
The bladder carcinogenicity of the urine and milk from cows fed bracken
fern has been assessed by the pellet implantation technique (see Section
4.3.3.5, Vol. I). Urine or milk from these animals was extracted with organic
solvents and the extract mixed with cholesterol for the preparation of the
pellets. Pellets containing these urine extracts introduced into the bladder
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Table XXXVI
Carcinogenicity of Some Bracken Fern Toxins3
Compound
Shikimic acid
Pterolactam
Pterosin B
Pteroside B
Ptaquiloside
(Aquilide A)
Species
and strain
Mouse, TF1
Rat , AC I
Mouse, Swiss
Rat, ACI
Rat , Wistar
Rat , Wistar %
Rat , Sprague-
Dawley
Route
i .p. or i .g .
oral
urinary bladder
implantation
oral
oral
oral
oral
Principal
organs
affected
Stomach,
haematopoiet ic
tissue
None
None
None
None
None
Mammary gland,
intestine
References
(3)
(73)
(50)
(50)
(16,
(16,
(71,
70)
70)
72)
aSee Table XXXIII for structural formulas.
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induced carcinomas in mice (68) and hemangiomatous lesions in calves, dogs and
rats (67). Pellets prepared with milk extracts caused bladder carcinomas in
10 of 24 mice; only 3 of 19 mice treated with control pellets (prepared from
milk of cows fed a normal diet) bore these tumors (29). Further evidence that
carcinogenic substance(s) is/are present in the milk of cattle consuming
bracken fern stems from the findings that 9 of 34 rats fed whole milk and 11
of 56 rats fed freeze-dried powdered milk from cows ingesting bracken fern
developed carcinomas of the urinary bladder, intestine or kidney (29).
When guinea pigs (21, 47) and rats (45, 54, 56, 57, 61) were given a
basic diet supplemented with bracken fern or when rats (61, 66) were admini-
t ^
stered cold or hot water extracts of bracken fern as drinking water, signifi-
cant incidences of urinary bladder neoplasms were found. Pamukcu et al. (52)
induced urinary bladder carcinomas in Swiss albino female mice by implantation
of pellets containing bracken fern into the bladder. Urinary bladder tumors
were also induced in 4 of 15 ICR strain mice fed powdered bracken fern in a
diet (25%) for 20 weeks, following surgical implantation of a glass bead into
the bladder. No such tumors were observed in a group of mice fed the bracken
fern diet without a glass bead in their bladder and in a control group bearing
a glass bean implant and fed a normal diet (51).
In addition to bladder cancer, mice and rats exhibited tumors at other
localizations following bracken fern administration. Lymphatic leukemia and
pulmonary adenomas have been reported in Swiss mice fed a bracken fern diet
(33%) every other week for a total period of 60 weeks (49). High incidence of
lung tumors was also induced in Swiss mice (21) and in dd strain mice (50) fed
bracken fern. Several studies have demonstrated that bracken fern induces a
significant incidence of intestinal tumors in rats of various strains (see
Table XXXV); spontaneous intestinal neoplasms are only seldom encountered in
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rats. It appears that feeding of bracken fern to rats for short duration
induces only intestinal tumors, whereas long-term feeding produces tumors of
the intestine and the bladder simultaneously. No difference in intestinal
tumor incidence was found between conventional and germ-free Wistar female
rats, suggesting that microflora of the gut may not play an important role in
bracken fern carcinogenesis (64). The carcinogenicity of processed bracken
fern treated with boiling water containing wood ash, sodium bicarbonate or
sodium chloride has been tested in ACI rats. Thus, the plant which has under-
gone processing for human food can still possess weak carcinogenic activity
(59). Bracken fern powder which has been stored for 1 to 2 years induces
intestinal tumors in ACI rat's7 although with a lower incidence and a longer
latent period, suggesting that the carcinogenic activity of bracken fern is
reduced by long-term storage (63). Recently, Hirono et al. (66) noted, in
addition to ileal and urinary bladder tumors, a 87% incidence of mammary
tumors in 15 female CD strain rats given a diet containing 30% bracken fronds
for up to 260 days. Furthermore, these authors (74) observed bile duct pro-
liferation and hyperplastic nodules in the liver in two strains of rats fed a
diet containing bracken fern or water extract of the plant.
Several other animal species have been found to be susceptible to the
carcinogenic action of bracken fern. Sheep and young hamsters fed bracken
fern diet developed gastrointestinal neoplasms (13, 21). In the sheep, both
intestinal and colonic adenocarcinomas were observed after feeding on bracken
fern pellets for 8 months (21). In the hamster, adenocarcinoraas were induced
predominantly in the cecum and ileum (13, 21). When 34 Japanese quail
(Coturnix coturnix japonica) received anethanol extract of dried bracken fern
mixed with their normal diet for the first 5 months after hatching, 27 of the
birds had adenocarcinomas of the intestine and colon which were not seen in
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the controls (21, 46). The Egyptian toad (Bufo regularis) is an effective
model for detecting the carcinogenicity of bracken fern. Seven ileal adeno-
carcinomas, 16 hepatomas and 6 tumors in the kidney due to metastases of the
hepatomas were noted in 18 of 98 toads following forced feeding with bracken
fern (10 mg/50 g body weight), once a week for only 20 weeks. Such lesions
were not seen in 100 control toads (48).
5.3.2.1.3.3 CARCINOGENICITY OF BRACKEN FERN TOXINS
Shikimic acid. In 1971, Leach, Barber and Evans (27) reported the isola-
tion of an active principle from bracken fern, carcinogenic to mice by i.p.
injection. Chemical analysis showed that the isolated compound is shikimic
acid (1). Following single i.p. injections (1-20 mg) or oral administration
of 100 mg aqueous shikimic acid to 14 weanling TF1 strain mice, 6 glandular
stomach tumors, 3 reticulum cell leukemias, 1 lymphocytic leukemia and 1 pul-
monary adenoma were found in 9 animals. None of the 57 control mice displayed
these tumors (1). Shikimic acid is also active in the BHK21 cell transforma-
tion assay (36). However, when shikimic acid was given to a group of ACI
strain rats in the diet (0.1%) for 142 days, no tumors were induced (73).
Furthermore, shikimic acid is the precursor in the biosynthesis of aromatic
ring compounds in a wide variety of plants. Thus, shikimic acid is not con-
sidered to be the principal bracken fern carcinogen.
Pterolactarn. The carcinogenic potential of pterolactam, a novel
5-membered lactam isolated from bracken fern, has been studied in mice and
rats. No significant neoplasms were found in 30 female Swiss mice which
received pellets containing pterolactam implanted surgically into the urinary
bladder. The compound also failed to exhibit carcinogenic effects when fed or
intragastrically administered to groups of young ACI strain rats (50).
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A structurally-related chemical, caprolactam (2-oxohexaraethyleneimine),
used in the production of nylon-6, was also found noncarcinogenic in rats and
mice under the study conditions of the U.S. National Toxicology Program (75).
Pterosin B and Pteroside B. These two major indanones are present in
abundance in the fronds and rhizomes of bracken fern. Pterosin B, pteroside B
or fractions of bracken fern extracts containing various other pterosins and
pterosides were given to groups of Wistar rats in the diet for up to 205
days. The daily doses of pterosin B (4 mg) and pteroside B (10 mg) were each
equivalent to about twice the level contained in a carcinogenic dose of dried
bracken fern. Nonetheless, no neoplasms developed in the rats (16, 70).
t- >
Phenindione (2-phenyl-l,3-indandione), an anticoagulant structurally related
to pterosins, was also non-carcinogenic under similar study conditions (70).
Ptaquiloside (Aquilide A). Ptaquiloside (aquilide A) is structurally
related to the illudins, a group of anti-cancer agents. This novel toxin was
isolated from bracken fern independently by Niwa e.t_ _al_. (6) and by van der
Hoeven and coworkers (7, 8) and was named by these two groups ptaquiloside and
aquilide A, respectively. This compound is not only a potent genotoxic agent
(see section on Mutagenic Effects), but it is also a strong carcinogen. In
vitro transformation assays (8) have shown that ptaquiloside induces type III
transformed foci in C3H 10T,i^ cells. When a fraction of the boiling water
extract of bracken fern containing ptaquiloside was given to 7 female CD rats
in the diet for 133 days, all animals developed mammary and intestinal tumors;
in 5 of them, urinary bladder neoplasms were also found (71). Similar results
were obtained in subsequent studies using purified ptaquiloside. High inci-
dences of mammary adenomas (100% and 91%) and ileal adenocarcinomas (57% and
91%) were induced in two groups of female CD rats administered ptaquiloside
(100-200 mg/kg body weight) once or twice a week for 8-9 weeks. Although
220
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urinary bladder tumor was observed only in one rat, preneoplastic hyperplasia
of the urinary bladder mucosa was present in 15 of 21 treated animals (71,
72). These are the findings which led to the conclusion that ptaquiloside
represents the carcinogenic principle of bracken fern.
5.3.2.1.3.4 MODIFICATION OF CARCINOGENESIS
Hirono et al. (76) have reported that ingestion of bracken fern enhances
the induction of tumors of the upper alimentary tract by N-propyl-N-nitroso-
urethan in ACI rats. On the other hand, various chemicals have been shown to
inhibit the carcinogenicity of bracken fern. For instance, administration of
nicotinamide (0.5%) or phenothiazine (0.2%) in the diet decreases the tumori-
r ^
genieity of bracken fern toward the intestine and urinary bladder by about 40-
50% (55, 57). Similarly, a 25-30% reduction in the incidence of intestinal
neoplasms induced by bracken fern was noted in rats fed a bracken fern-
containing diet supplemented with disulfiram, calcium chloride or butylated
hydroxyanisole; dietary calcium chloride or polyvinyl pyrrolidone supplementa-
tion suppressed the bracken fern-induced urinary bladder tumorigenesis by
about 80% (56). However, a significantly higher incidence of urinary bladder
carcinomas, however, was induced in rats fed a diet containing bracken fern
and thiamine hydrochloride compared to rats fed a bracken fern-containing diet
without thiamine (55) .
5.3.2.1.4 Metabolism and Possible Mechanisms of Action
Among the chemicals isolated from bracken fern, shikiraic acid, tannin,
quercetin, kaempferol, pterolactam, pterosins, pterosides and ptaquiloside
have been suspected to account for the carcinogenic action of the plant. How-
ever, the data available show that pterolactam, pterosins, and pterosides are
not carcinogenic or mutagenic (see previous Sections). Tannin, quercetin,
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ptaquiloside, and other phenolics and flavonoids present in bracken fern may
play a role in the carcinogenic activity; the metabolisms and possible
mechanisms of action of these substances are discussed in Sections 5.3.2.6.2
and 5.3.2.6.3.
The metabolism of shikimic acid has been studied quite extensively by
Brewster, Jones and Parke (77-79). Figure 9 shows the major metabolic path-
ways of shikimic acid in the rat. After oral administration of shikimic acid
to rats, benzpic acid, hippuric acid, 3,4,5,6-tetrahydrohippuric acid, hexa-
hydrohippuric acid and cyclohexylcarbonyl-^3-D-glucoronide were identified as
principal metabolites in the urine (79). These metabolites are produced by an
t- %
initial conversion of shikimic acid to cyclohexanecarboxylic acid by the
intestinal flora followed by further metabolism in rat tissues (77, 78).
Since none of the metabolites of shikimic acid exhibits, any mutagenic and
cell-transforming activity (36), the metabolism of shikimic acid probably
represents a detoxification process. On the other hand, since shikimic acid
itself is an **>fi -unsaturated carboxylic acid, it may react with cellular
nucleophiles (especially amino groups) by a Michael-type addition similarly to
acrylic acid (see Appendix I).
The metabolism of ptaquiloside has not been investigated. However, on
the basis of its chemical reactivity under acidic and alkaline conditions (see
Section 5.3.2.1.2.1), it was speculated that ptaquiloside may be inactivated
to pterosin B in the stomach, whereas in the intestine and in the urine of
herbivorous animals some activation of ptaquiloside to aquiline A may occur
and this results in intestinal and urinary bladder tumorigenesis (8). The
findings that bracken fern-induced intestinal adenocarcinomas occur predomi-
nantly in the ileal region, where the pH level is the highest, appears to
support this hypothesis. It also explains the observations that treatment
222
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CONHCH2COOH
3, 4, 5, 6 - Tetrahydrohippuric
acid
HO
OH
Shikimic acid
COOH
COOH
Cyclohexanecarboxylic
acid
Beruoic acid
CONHCH2COOH
Hippuric acid
o
it
C-0-C6H906
CONHCH2COOH
Cyclohexylcarbonyl- Hexahydrohippuric
- ft- D -glucuronide acid
Fig. 9. Major metabolic pathways of shikimic acid in the rat.
-------
with alkali reduces the carcinogenic activity of bracken fern (59) and that
ptaquiloside is more potently mutagenic toward Salmonella under alkaline con-
ditions (8). Aquiline A, the proposed reactive intermediate, may exert its
carcinogenic and mutagenic action by virtue of the potential reactivity of the
vinyl carbonyl and/or the cyclopropyl ring moieties in the molecule.
5.3.2.1.5 Environmental Significance
Bracken fern is an ubiquitous plant in the temperate and midtropic zones
of the world. It grows well in moist, shady soils in a variety of habitats
ranging from sea level to hilly pasture lands 2,000 feet or higher. As a
forage contaminant, bracken ,f«rn has been responsible for many cases of cattle
poisoning and death in various parts of the world, particularly in Scotland,
Australia, New Zealand, Turkey and Brazil. A close association between the
geographic distribution of bracken fern and the high rate of urinary bladder,
intestinal and esophageal tumors in cattle and sheep in these countries has
been observed (rev. in 13, 80).
In Japan, Canada and the northeastern United States, young fronds of
bracken fern are consumed by humans as a food delicacy or salad greens. More-
over, indirect human exposure takes place through consumption of meat and milk
(or dairy products) from animals grazing on bracken fern. For this reason,
the marked regional prevalence of some forms of human cancer, such as
esophageal and stomach cancers in Japan and North Wales, has been suspected to
be linked to the exposure of bracken fern toxins. An epidemiological study of
the cancer incidence of people living in a mountainous district of central
Japan, where considerable quantities of bracken fern are consumed, revealed
that daily intake of bracken fern does indeed increase significantly the risk
to esophageal cancer. The relative risk is even higher for those who also
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consume hot tea gruel ("chagayu" in Japanese) and smoke cigarettes daily
(81). The role of bracken fern toxins in the genesis of other human cancers
still awaits further investigation.
Among various toxins isolated from bracken fern, shikimic acid, tannin
and several flavonoids also occur at variable amounts in many different
plants. For instance, among the 268 plant species examined, 158 (many of
which are food plants) have been found to contain shikimic acid in various
parts of the plants (15). Trace amounts of shikimic acid (up to 0.193% fresh
weight) have also been detected in 56 of 83 species of Angiosperm leaf samples
from Japan (82). The distribution of tannin and flavonoids in plants is dis-
cussed in Section 5.3.2.6.2 and in Section 5.3.2.6.3.
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SOURCE BOOKS AND MAJOR REVIEWS OF SECTION 5.3.2.1
1. Bryan, G.T., and Pamukcu, A.M.: Sources of Carcinogens and Mutagens in
Edible Plants: Production of Urinary Bladder and Intestinal Tumors by
Bracken Fern (Pteridium aquilinum). In "Carcinogens and Mutagens in the
Environment" (H.F. Stich, ed.), Vol. I, CRC Press, Boca Raton, Florida,
1982, pp. 75-82.
2. Pamukcu, A.M., and Bryan, G.T.: Bracken Fern, a Natural Urinary Bladder
and Intestinal Carcinogen. Jto "Naturally Occurring Carcinogens-Mutagens
and Modulators of Carcinogensis" (E.C. Miller, J.A. Miller, I. Hirono,
T. Sugimura and S. Takayama, eds.), Japan Sci. Soc. Press, Tokyo/Univ.
Park, Baltimore, 1979, pp. 89-99.
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Searle, ed.), ACS Monograph 173, American Chemical Society, Washington,
D.C., 1976, pp. 690-700.
230
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4. Hirono, I.: CRC Grit. Rev. Toxicol . 8, 235-277 (1981).
5. Bohm, B.A.: Chem. Rev. 65, 435-466 (1965).
6. Stavric, B., and Stoltz, D.R.: Food Cosmet. Toxicol. 14, 141-145
(1976).
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