CURRENT AWARENESS DOCUMENT
NITROSAMINE CONGENER ALKYLAZOXYMETHANOL-DERIVED ALKYLATING AGENTS:
CYCASIN AND RELATED COMPOUNDS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by:
Yin-Tak Woo, Ph.D., D.A.B.T.
David Y. Lai, Ph.D.
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.
Extradivi sional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5.3.2.2 Cycasin and Related Compounds
5.3.2.2.1 Introduction
Cycasin is a member of a group of naturally occurring azoxyglycosides
produced by the palm-like cycad plants. Belonging to the ancient family
Cycadaceae of the Gymnospermae, cycads are thought to represent an inter-
mediate form in the phylogenetic evolution of plants from ferns to flowering
plants. Currently, there are about 100 species of cycads, identiifed as
members of 9 genera (Bowenia, Ceratozamia, Cycas, Dioon, Encephalartos,
Macrozamia, Microcycas, Stangeria, and Zamia)*, growing in tropical and sub-
tropical regions around the world. Various parts of these plants were and
i •%
still are used as a source of food in some parts of the world, particularly
during periods of natural disasters.
The historical development of early cycad research was eloquently
described in a 1963 review by Whiting (1). The toxicity of cycads has long
been known by the natives who have developed elaborate procedures to remove
the poisonous substances. Explorers and early settlers were frequently the
victims of toxic effects of cycads as they experimented with these unfamiliar
native foods. In 1770, members of Captain Cook's crew were reported to have
become violently ill after consuming cycad (Cycas media) nuts during their
voyage to Australia. Outbreaks of cycad poisoning and heavy losses of live-
stocks occurred in Australia between the 1880's and the 1930"s as the rapid
growth of the sheep and cattle industry led to the expansion of grazing into
*Some taxonomists separate an additional genus (Lepidozamia) from Macrozamia
and classify cycads into three families: Cycadaceae (1 genus, Cycas);
Stangeriaceae (1 genus, Stangeria); and Zamiaceae (includes all the other
genera).
232
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the natural habitat of cycads. Scientific interest in cycad research began in
the late 19th century, but it was not until 1941 when Cooper (2) succeeded in
isolating a glycoside from Macrozamia spiralis. The compound, named macro-
zamin, was subsequently structurally identified as methylazoxyraethanol-ft-D-
primeveroside (3, 4). Soon afterwards, Riggs (5, 6) and Nishida et al. (7)
isolated cycasin from cycad plants found in Guam (Cycas circinalis L.) and
Japan (C. revoluta Thunb) and identified its structure as raethylazoxymethanol-
ft-D-glucoside.
During the mid-1950's, clinical observations of the natives of Guam (the
Chamorros, a Malayo-Polynesian group) showed an unusually high incidence of a
neurological disease diagnosed^ as amyotrophic lateral sclerosis (8). Based on
evidence collected from reports of neurological disorders in cycad-poisoned
catties and sheeps (1), Whiting suggested a possible link between native
consumption of cycad and the disease. An attempt to experimentally prove such
a link using rats was unsuccessful. Instead, however, Laqueur _et_ _al_. (9)
found that rats fed a crude meal derived from cycad (C. circinalis L.) seeds
from Guam developed a variety of tumors. Subsequent studies using purified
cycasin and cycasin-free cycad meal (see Section 5.3.2.2.3.1) established
cycasin as the active carcinogenic principle in the crude cycad meal. Methyl-
azoxymethanol, the aglycone of cycasin, has been shown to be the proximate
carcinogen of the azoxyglycoside (see Section 5.3.2.2.3.2). Products derived
from a variety of other cycad species (including the Encephalartqs spp. which
produce macrozamin) also display carcinogenic properties. In addition,
cycasin is teratogenic, when given to pregnant rats, and neurotoxic when given
to various newborn animals (see Section 5.3.2.2.2.2). These findings have
stimulated a wave of interest in cycad research culminating in a succession of
six conferences between 1952 and 1972. The proceedings of three of these con-
233
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ferences were published (10-12). Cycasin and related compounds have continued
to attract considerable scientific interest which is reflected by the large
number of recent reviews (13-19).
5.3.2.2.2 Physicochemicai Properties and Biolo'gical Effects
5.3.2.2.2.1 PHYSICAL AND CHEMICAL PROPERTIES
The physical and chemical properties of cycasin and related compounds
were extensively studied by several groups of investigators in Australia and
Japan (2-7, 20-25). The chemical structures and some physicochemicai
properties of these compounds are given in Tables XXXVII and XXXVIII, respec-
tively. Cycasin is soluble in water or aqueous ethanol, sparingly soluble in
i ^
pure ethanol, and insoluble in most organic solvents. The ultraviolet absorp-
tion spectrum of cycasin shows a pronounced maximum around 217 nm, and an
inflexion at 275 nm. The infrared spectrum displays a strong absorption band
around 1,540 cm , characteristic of aliphatic azoxy compounds. Cycasin is
readily hydrolyzed at 100°C with 0.1 N HC1, yielding methanol, formaldehyde,
nitrogen and glucose. Mild alkali treatment also leads to decomposition,
yielding formic acid, cyanide, nitrogen, ammonia and methylamine (7). Essen-
tially identical physicochemicai properties have been reported for other
azoxyglycosides such as macrozamin (4, 7), and the neocycasins A, B, C, and E
(20-23).
Methylazoxymethanol (MAM), the common aglycone of all the above azoxy-
glycosides, is a colorless liquid with an amine-like odor; it has a density of
1.208 and boils at 51°C under reduced pressure (0.56 mm Hg). It is totally
miscible with water and aqueous ethanol, and is slightly soluble in chloroform
and ether. Both pure and aqueous solutions of MAM are unstable at room
temperature; approximately 12.5% of pure MAM and 21.3% of aqueous MAM decom-
234
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XXVII
Structural Formulas of Cycasin and Related Compounds
0
t
CH3-N=N-CH2OH
Methylazoxymethanol
(MAM)
0
t
CH3-N=N-CH2OR
MAM Acetate (R=CH3CO-)
MAM Benzoate(R=C6H5CO-)
CHOH
00-CH2-N=N-CH
OH
Cycasin
COOH
0 0-CH2-N=N-CH3
0
HO
OH
MAM-/3-D-glucosiduronic
acid
Q
rO-CH,
HO
HO HO
0
]—0V0-CH2-N=N-CH3
OH
OH
Macrozamin
CH2OH
Q
0
I
HOH2C
HO
0.
H
p-CH2-N=N-CH3
O OH
OH
Neocycasin A
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Table XXXVIII
Physicochemical Properties of Cycasin and Related Compounds
Compound
Cycasin
Neocycasin A
Neocycasin B
Neocycasin E
Macrozamin
Methylazoxy-
methanol (MAM)
MAM acetate
MAM benzoate
MAM- B-D-
glucosi-
duronic acid
Synonyms and abbreviations
MAM- B-D-glucoside; B -D-
glucosyloxyazoxyme thane
3-0- B -D-glucosylcycasin;
B-laminar ibosyloxyazoxy-
methane; MAM- B -laminar iboside
6-0- B -D-glucosylcycasin;
B-gent iobiosyloxyazoxy-
methane; MAM- B -gent iobioside
4-0- B -D-glucosylcycasin ;
B-cel lobiosyloxyazoxy-
methane; MAM- B -eel Iobioside
6-0- B -D-xylosylcycasin ;
B-pr iraeverosyloxyazoxy-
methane; MAM- B -pr imeveroside
Hydroxymethylazoxymethane
MAM Ac; MAMA
MAMB
MAM glucuronide
m.p. b.p. Optical rotation
144- — (odn8 = -41-3°
145°C U
162- - [o(]*9 = -35.1°
163°C °
173- ~ l°Cln* = -37.6°
174°Ca °
156- -- tod15 = -29.2°
158°C D
199- ~ [
1
IR~Amax References
1,540 cm"1 (7)
1,537 cm"1 (20)
(21)
(23)
1,538 cm"1 (4,7,
26)
1,515 cm"1 (25)
1,522 cm"1 (25)
1,517 cm"1 (25)
1,540 cm"1 (27)
a
Ac e t a t e f o rm.
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pose after 5 and 5.6 hrs, respectively (25). Heat, acid and alkali greatly
accelerate the rate of decomposition. Approximately 75% of MAM decomposes in
aqueous solution by heating at 75°C for 30 min, and close to 100% when heated
at 100°C for 10 min (25). Under physiological conditions (37°C; pH 7-8), the
half-life of MAM in aqueous solution is about 11.5 hours (28). The half-life
is considerably shorter in alkaline solutions, decreasing to 2.8 hours at pH
10. The ester of MAM with acetic acid has similar physical properties as MAM
but is considerably more stable; MAM acetate can withstand decomposition in
aqueous solution when heated at 75°C for 30 minutes (25). The benzoic acid
and ft-D-glucosiduronic acid esters of MAM are both solid at room temperature,
with melting points of 66°C ,a^d 109-110°C, respectively (25, 27).
5.3.2.2.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY
Toxic it y . The toxicology of cycasin and cycad products was the subject
of several extensive reviews (1, 16, 29) and several international conferences
(10-12). The acute LDcg data on cycasin and related compounds are summarized
in Table XXXIX. Cycasin is acutely toxic to rodents when administered by the
oral route, but nontoxic when injected int raperitoneally (9, 25, 31, 33),
suggesting that enzymatic hydrolysis by the intestinal bacterial flora is
required for the toxic action of cycasin. This is supported by the finding
that both the aglycone of cycasin, methylazoxymethanol (MAM) and MAM acetate
are toxic in rats by intraperitoneal injection producing essentially the same
toxic effects as orally administered cycasin (25, 36). The principal target
organs of the acute and subchronic toxic action of cycasin and MAM are the
liver, and the brain (in developing animals). The hepatotoxic effects
observed in rats given cycasin or MAM acetate include loss of cytoplasmic
basophilia, focal cellular necrosis, pyknosis of nuclei and cytoplasmic
eosinophilia, progressing eventually to hemorrhagic cent r ilobular necrosis
(13, 16, 36).
235
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Table XXXIX
Acute Toxic icy of Cycasin and Related Compounds
Compound
Cycasin
-
-
Macrozamin
Methylazoxymethanol (MAM)
MAM Acetate
MAM- ji-D-glucosiduronic acid
Species and Route
Mouse, oral
Rat , oral
Hamster, oral
Guinea pig , _oral
Rabbit , oral
Fish, oral
Rat , oral
Rat, i.p.
Mouse, i.v.
Rat , i .v.
Rat , i.p.
Chick embryo0
Rat , i.p.
LD5Q (mg/kg)
500; 1,000
270; 562
<250
. <20; L..OOO. .
30
LC5Q = 20 ppma
218
LDlQ = 35b
25-30
35-50
90
405 /<,!
>1,000
Reference
(30,
(32,
(30)
_ (30,
(30)
(34)
(26)
(25)
(35)
(35)
(36)
(37)
(27)
31)
33)
31)
aLethal concentration causing 50% kill.
Lowest lethal dose.
clnjected into yolk sac of the egg.
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Newborn animals are extremely sensitive to the neurotoxic effects of
cycasin and MAM acetate. Neonatal exposure of mice (38, 39), rats (40-43),
hamsters (40, 44-46), ferrets (40, 47, 48), rabbits, cats and dogs (40) to
cycasin or MAM acetate causes defects during the maturation of the cerebel-
lum. The most notable abnormalities in rodents include misalignment or dis-
orientation of Purkinje cells, deletion of granule cells and cerebellar dys-
function (41, 42, 49). The locomotive activity of the afflicted animals may
be severely affected (43, 44, 48) resulting in gait disturbances and ataxia.
In addition to cerebellar malformation, severe atrophy of the retina has been
observed in hamsters (46) or rats (50) treated with MAM acetate and in mice
and rats treated with cycas^n^(30) within a few days after birth.
Besides experimental animals, farm animals grazing on leaves or seeds of
Cycas or Macrozamia plants (1) and humans consuming improperly prepared food-
stuffs derived from Cycas plants (1, 51) were reported to develop various
symptoms of acute intoxication. Outbreaks of cycad poisoning of cattle and
sheep occurred frequently in Australia between 1879 and 1930 resulting in
considerable livestock losses. The most commonly observed acute symptoms were
severe gastrointestinal disturbances and partial paralysis of the hind legs
(rev. 1). Cycad plants have long been known to be poisonous by natives who
have developed methods of detoxifying cycad products. Cases of human poison-
ing and casualties usually occurred during periods of extreme food shortage or
as a result of lack of knowledge when improperly prepared cycad products were
consumed (1, 51). Hirono et al. (51) reported 17 cases of fatalities as a
result of ingestion of cycad products on Miyako Island (Okinawa, Japan). The
latent period for the appearance of symptoms was 12-24 hours. The majority of
the patients died within several to 20 hours after the first sign of intoxica-
tion. The symptoms which occurred suddenly included headache, stomachache,
236
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nausea, vomiting, convulsion and loss of consciousness. In most patients,
swelling of the liver was observed.
Mutagenicity. The mutagenicity of cycasin and related compounds has been
tested in a variety of systems ranging from bacteria, yeasts, plants,
Drosophila, cultured mammalian cells to whole mammals. Table XL summarizes
the results of the Ames Salmonella tests of these compounds. This subject has
been reviewed in-depth by Morgan and Hoffman (18,_ 19). __With one, except ion
(56), cycasin was consistently nonmutagenic in in vitro studies using bacteria
(52-55), yeasts (64) and cultured mammalian cells (65), regardless of the
presence or absence of a metabolic activation system of mammalian hepatic
origin (S-9 mix). However,'c^rcasin is readily rendered mutagenic by enzymatic
deglucosylation using p-glucosidase (52, 54, 58, 65), fecalase from human
fecal extract (55) or rat intestinal flora (64), suggesting that the release
of methylazoxymethanol (MAM) is a requisite for the expression of the muta-
genic activity of cycasin. This is supported by the findings that neocycasin
A and MAM-/3-D-glucosiduronic acid (the glucuronide of MAM) are also nonmuta-
genic per se but are activated by incubation with fecalase or Q-glucuroni-
dase, respectively, which release MAM (27, 54, 55). In host-mediated assays
using Salmonella typhimurium G46 strain as an indicator organism, cycasin is
mutagenic when administered orally, but inactive when administered paren-
terally. The mutagenicity of cycasin is abolished by pretreating the host
(mouse) with an antibiotic that kills the intestinal bacterial flora (57).
Among higher plants, cycasin displays mutagenic activity in beans (Phaseplus
vulgaris L.) inducing chlorophyll mutations and morphological alterations
(66), and towards root cells of onion (67) or Zamia integrifolia (68) causing
chromosome aberrations. Both bean seeds and Zamia plants contain
-glucosidase. In contrast, mutagenicity tests using Drosophila yielded
237
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Table XL
Mutagenicity of Cycasin and Related Compounds in the Ames Test
Compound
Cycasin
Neocycasin A
Mac rozamin
Methylazoxymethanol
With or without S-9
- (52-55); + (56)b
- (55)
- (55,56)
+ (52,54,58)
Preincubat ion
3-glucosidase fecalese3
+ (52,54) + (55)
+ (55)
- (55)
>
with
3-glucuronidase mediated
+ (57)c
(MAM)
MAM Acetate
(53,54,59-63)
MAM- 3-D-glucosiduronic - (27,54,55)
ac id
- (55)
(27,54)
(59,62)
aContains both 3~glucosidase and B~glucuronidase activity.
Positive in the presence of S-9.
cNegative if the host was pretreated with an antibiotic.
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negative results (69, 70) consistent with the failure to detect /3-glucosidase
activity in homogenates of Drosophila (69). In experimental animals, orally
administered cycasin induces strand breakage in rat liver DNA (71) and causes
fragmentation in mouse liver DNA (72).
Consistent with the conclusion that MAM is the proximate or ultimate
mutagen of cycasin, MAM and MAM acetate display mutagenic activity in a
variety of test systems. In most cases, an exogenous metabolic activation
system is not required for mutagenicity. Using the Ames tests, numerous
investigators (52-54, 57-60, 62) have shown that MAM or MAM acetate induces
base-pair substitution mutations in the absence of metabolic activation.
Inclusion of S-9 mix has a variable effect ranging from enhancement of muta-
genicity (54) to activation to a frameshift mutagen (63, 73) or to reduction
of mutagenicity (60, 73). Methylazoxymethanol also induces base-pair substi-
tution mutation in Escherichia coli WP2 trp~ in the absence of metabolic
activation (74). In yeasts, MAM acetate is both mutagenic and recombinogenic
(75-78). The yeast strains tested include Saccharomyces cerevisiae D3, D7,
T2, and XV185-14C. With one exception (75), exogenous metabolic activation
system is not required for expression of optimal activity of MAM acetate. In
contrast to the lack of mutagenicity of cycasin in Drosophila, MAM acetate is
clearly mutagenic in the fruit fly (69, 70, 79), indicating that the absence
of (3 -glucosidase activity accounts for the lack of mutagenicity of cycasin in
the fruit fly.
Evidence indicating the mutagenicity of MAM or MAM acetate in mammalian
cells is abundant. In in vitro studies, these compounds produce a variety of
genetic damages as indicated by the induction of mutation (80-83), chromosome
aberrations (84, 85), sister chromatid exchanges (86-88), and unscheduled DNA
synthesis (89-91). The ability of MAM acetate to induce mutation in V79
238
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Chinese hamster cells is enhanced by tumorigenesis promoters, such as phorbol
esters, and cocarcinogens, such as C^ to C^ linear alkanes (80). The comuta-
genic activity of these compounds correlates with their promoting or cocar-
cinogenic activity. In in vivo studies, MAM acetate treatment was shown to
cause DNA damages (92-94), chromosome aberrations (95, 96) and an increase in
the frequency of sister chromatid exchanges (88) in liver, bone marrow, spleen
and other cells.
Teratogenicity. Methylazoxymethanol (MAM) or MAM acetate is teratogenic
in at least three animal species, with the central nervous system being the
principal target tissue. Spatz et al. (97) demonstrated first the terato-
genicity of MAM using the gofcden hamster. A single intravenous injection of
20-23 rag/kg MAM to pregnant hamsters on the 8th day of gestation led to mal-
formations of the brain, eye and extremities in all of the living fetuses
examined on the 12th day of gestation. The gross malformations observed
include hydrocephalus, microcephalus, cranioschisis, exencephaly, spina
bifida, rachischisis, anophthalmia, microophthalmia, and oligodactyly. No
attempt was made to observe the development of the malformed fetuses beyond
the 12th day of gestation. The finding of malformations of the brain and the
spinal cord in this study suggests rapid action of MAM on embryonic develop-
ment, since the neural tube of hamster fetus closes by the 9th day of gesta-
tion. A preliminary histological study of MAM-treated hamster fetuses by
Laqueur (see 16) indicated necrosis of cells in the embryonic region known to
be involved in the closure of the neural tube.
Cerebral malformation is the principal teratogenic effect of MAM or MAM
acetate in the rat and in the ferret. Studies by various investigators (16,
98-101) have consistently shown that a single administration of 20-30 mg/kg
MAM or MAM acetate to pregnant rats of various strains (Fischer, Osborne-
239
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Mendel, Sprague-Dawley, Long-Evans) on the 15th day of gestation induces a
high incidence of microencephaly in the offspring. The earliest recognizable
lesion (necrosis of undifferentiated cells in the region destined to produce
the final cerebral cortex) leading to microencephaly was visible within 24
hours after administration of MAM (see 16). Neurochemical studies (102-104)
revealed that MAM is selectively cytotoxic to the cortical Y^arainobutyric
acid (GABA)-containing neurons resulting in a severe loss of GABAergic
neurons. In contrast, the noradrenergic neurons (102) and oligoendroglia
(105) are minimally or not affected. Injections of MAM acetate (15 mg/kg)
into pregnant ferrets on days 27-32 of gestation (the full gestation period of
this species is 42 days) result in malformations of the cerebral hemisphere
(microencephaly, lissencephaly, etc.), whereas similar treatment in later
periods (days 37-42) produces malformations of the cerebellum (40, 47, 48).
As adults, the ferrets with cerebral malformation (particularly those with
lissencephaly) display learning difficulties, whereas those with cerebellar
abnormalitites show no cognitive deficits despite being ataxic (48). It has
been suggested that the MAM-induced lissencephalic ferrets may be a useful
experimental model for the study of lissencephaly, which also occurs in humans
as a severe birth defect with unknown etiology.
Besides prenatal exposure, perinatal or neonatal exposure to cycasin or
MAM acetate can also cause abnormalities in the development of the nervous
system in several species. The most notable effects are malformation of the
cerebellum and atrophy of the retina (see above under Toxicity).
240
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5.3.2.2.3 Carcinogenicity and Structure-Activity Relationships
5.3.2.2.3.1 CARCINOGENICITY OF CYCASIN AND CYCAD PRODUCTS
Since the first report in 1963 of carcinogenicity of crude cycad (Cycas
circinalis L.) meal in rats, cycasin or cycad products have been found to be
carcinogenic in 7 of 8 animal species tested. The cycad products thus far
examined include nuts, kernel, husk, leaves, extract or flour derived from 10
different species of cycad plants belonging to 3 genera that grow in Guam,
Japan, Australia and Central, East and South Africa. Most of the earlier
carcinogenicity studies on cycasin and cycad products have been reviewed by
Laqueur and Spatz (13, 16, 29) and by Hirono (17). The major findings of
these and the more recent studies are summarized in Table XLI. Cycasin has
also been used in several syncarcinogenesis studies which are discussed in
Section 5.3.2.2.3.4.
Studies by Laqueur, Spatz, Hirono and various other investigators have
clearly established cycasin as a potent carcinogen in the rat. The carcino-
genicity of cycasin is dependent on the route of administration, the dosing
regimen and the age of the animals. In virtually all studies using adult
animals, the oral route is the only effective route of administration. As
substantiated by the evidence analyzed below and in Section 5.3.2.2.3.2,
enzymatic deglucosylation by the intestinal bacterial flora to the aglycone,
methylazoxymethanol (MAM), is the requirement for the carcinogenicity of
cycasin and cycad products. The principal carcinogenicity target organs of
cycasin in the rat are the liver, the kidney and the intestines. Liver tumors
are induced principally by long-term administration of relatively low doses of
cycasin (9, 32, 106, 107, 109, 114, 115), whereas short-term administration of
higher doses of cycasin predominantly induces kidney tumors (33, 110, 112-
241
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Table XLI
Care inogenicity of C yea sin and Cycad Products in Various Animal Species
Species and Strain
Cycad Products Tested*
Route
Principal Organs Affected
References
Rat, Osborne-Hendel
or Sprague-Dawley
Rat, Osborne-Mendel
Rat, Sprague-Dawley
Rat, Sprague-Dawley
Rat, Sprague-Dawley
(germ-free)
Rat, Wistar
Rat, ACI
Rat, Sprague-Dawley
Rat, Osborne-Mendel
Rat , unspecified
Crude eye ad (£. circinalis L.)
meal or cycasin
Guamanian home-made eyead
(C. circ inalis L.) flour
Cycad (C. circinalis L.) husks
Cycad (£. circinalis L.) husks
Cycasin
Rat, Sprague-Dawley Cycasin
Rat, Osborne-Mendel Cycasin
Cycasin
Cycasin
Zamia (Z. debilis) leaves
Zaraia (Z. floridana) tubers
Flour from £. hildebrandt ii
nuts
oral Liver, kidney, intestines (9, 106, 107)
oral None (108)
oral Liver, kidney (109)
j
oral Kidney, liver (110)
oral ' None (106, 111)
oral Liver, kidney, intestines, (32, 112)
mammary gland
oral Kidney, intestines, liver, (33)
lung, brain
oral Kidney (113)
oral Testis (112)
oral None (110)
oral Kidney, liver, colon (16)
oral Liver, kidney, lung (114, 115)
-------
Table XLI (continued)
Species and Strain
Rat, Wistar
Rat, Fischer
(newborn)
Rat, Wistar
(newborn)
Mouse, C57BL/6
Mouse, C57BL/6
(newborn or
suckling)
Mouse , dd
(newborn)
Mouse , C57BL
Hamster, Syrian
golden
Hamster, Syrian
golden (newborn)
Guinea pig
Cycad Products Tested3
South African cycad
(Encephalartos spp.) >c
cones
Cycasin
Cycasin
Cycasin
Cycasin
Cycasin
Cycad (C. circinalis L.)
nut extract
Cycasin
Cycasin
Crude cycad (C. circinalis L.)
Route
oral
s .c .
s .c .
or i .p.
oral
s .c .
s.c .
skin
painting
oral
s.c .
oral
Principal Organs Affected
Kidney, liver
Kidney, liver, lung,
intestines, brain
Kidney, liver
Liver, lung, kidney (low
incidence)
Liver, hematopoietic system
Lung, liver
Liver , kidney
I
Liver, intestines,
hematopoietic system
Liver
Liver
References
(116,
(33)
(118)
(119)
(119,
(121)
(122)
(123)
(123)
(124)
117)
120)
meal
-------
p. 3.
Table XLI (continued)
Species and Strain
Rabbit
Chicken
Fish (Brachydanio
rerio)
Monkey (rhesus ,
cynomolgus or
African green)
Cycad Products Tested3
Crude cycad (C. revoluta)
extract
Cycad (C. circinalis L.) nut
husk or kernel
Cycad (C. circinalis L.) nut
meal or cycasin
Combination of cycad nut meal,
cycasin and synthetic
methylazoxymethanol acetate
Route
oral
oral
oral
oral
Principal Organs Affected
Liver
None
Liver
Liver, kidney
References
(125)
(126)
(34)
(127)
aThe genera of the cycad plants are: C. = Cycas; E. = Encephalartos; Z. = Zamia.
Known to contain macrozamin (methylazoxymethanol- fj-primeveroside)
cThe species identified include: E. umbeluziensis; E. villosus, E. lebomboensis, E. laevifolius and E.
lanatus.
Topical application to chemically induced skin ulcers.
-------
115). Intestinal tumors, which are almost exclusively located in the large
bowel (cecum, colon, rectum), appear to be less dependent on the duration of
exposure (29, 33, 107).
The dose-response relationship in the induction of tumors by cycasin has
been studied by Hirono £t__a_l_. (33). A single intragastric dose of 100 mg/kg
cycasin was sufficient to induce tumors in 4 of 13 rats. Two higher doses
(250 or 500 mg/kg) induced tumors in 100% of the treated animals whereas doses
higher than 750 mg/kg were acutely toxic (LD5Q = 562 mg/kg). The optimal dose
for tumor induction consistent with the greatest number of survivors was 250
mg/kg body weight. Continuous administration of low doses (4 mg/kg) of
cycasin failed to induce tumors in the liver, kidneys or intestines but
accelerated the development of tumors known to occur spontaneously (e.g. ,
mammary gland tumors in female Sprague-Dawley rats, testicular interstitial
cell tumors of male ACI rats) in rats (112). Hepatic tumors were induced in
Sprague-Dawley rats at a high incidence (80-90%) by continuous feeding of 10
mg/kg cycasin for the entire lifespan of the animals (32).
The carcinogenicity of orally administered cycasin in adult rats is
greatly dependent on the presence of intestinal bacterial flora. Laqueur et
al. (106, 111) showed that cycasin was not carcinogenic when administered to
"germ-free" rats. This gnotobiotic protective effect is due to the absence of
intestinal flora (such as Lactobacillus salivarius and Streptococcus fecalis),
which contain fi-glucosidase that can deglucosylate cycasin to MAM. Cycasin
ingested by germ-free rats is excreted quantitatively and unchanged in the
urine and feces (128, 129). The demonstration of potent carcinogenicity of
MAM and MAM acetate in both conventional and germ-free rats (see Section
5.3.2.2.3.2) confirms the indispensable, key role of ^-glucosidase in the
activation of cycasin.
242
-------
Studies with newborn rats further substantiate this mechanism. In con-
trast to the lack of carcinogenicity of parenterally administered cycasin in
adult rats, subcutaneous or intraperitoneal injection of cycasin to newborn
rats leads to the induction of a variety of tumors (33, 118). Consistent with
this, the enzyme assay of skin homogenates from fetal, newborn and adult rats
by Spatz (130) showed that neonatal rats (6 days before birth to 2 days after
birth) contain significantly high levels of ft-glucosidase activity; however,
the activity of the enzyme falls sharply as the rats reach adulthoo'd. Newborn
rats are highly susceptible to the carcinogenic effect of cycasin. In one
study (33), a single subcutaneous dose of 2.5 mg/rat was sufficient to induce
tumors in 46/55 rats, whereas in another study (118) a single intraperitoneal
> •>
or subcutaneous dose of 90 mg/kg cycasin induced tumors in 6/14 rats.
The cycad products which have been shown to be carcinogenic in the rat
are derived from Cycas circinalis L., (9, 106, 107, 109, 110), Zamia floridana
(16) and six different species of Encephalartos plants (114-117). Cycas
circinalis L. is indigenous in Guam; the carcinogenic azoxyglucoside it con-
tains has been identified as the glycoside of methylazoxymethanol, named
cycasin. A sample of home-made cycad flour from Guam was found to be cycasin-
free and was not carcinogenic in the rat (108). The processing procedure of
the natives can, apparently, remove cycasin and eliminate the carcinogenic
potential of the cycad product. Besides _C_. circinalis, rats fed diets con-
taining 2% or 3% ground Z. floridana tubers had tumor incidences of 12% and
60%, respectively. The organotropism and the histological types of tumors
induced by Z^. floridana are similar to those of cycasin (16). The chemical
structure of the carcinogenic azoxyglycoside(s) present has not been eluci-
dated. Dried leaves of Z. debilis have been tested by Hoch-Ligeti et al.
(110). Despite the presence of cycasin and macrozamin (at concentrations
243
-------
higher than those of cycad husks), Zamia leaves were not carcinogenic in the
rat. The reason for the inactivity is not clear. Various species of
Encephalartos plants have been found in Central, East and South Africa.
Mugera and Nderito (114) found that rats fed diets containing 5% or 10% flour
derived from E. hildebrandtii nuts for 6 to 10 months developed a variety of
tumors in the liver, kidneys and lungs; on short-term exposure (4-7 days),
however, the kidneys were the only affected organ (115). Tustin (116, 117)
fed rats the outer flesh and/or kernel of five species of South African cycad
plants (E. jimbeluziensis , E_. villosus, E^. lebomboensis, E_. laevifolius and _E_.
lanatus) and found them to be all carcinogenic, inducing mainly kidney and
liver tumors. The principal azoxyglycoside present in E. hildebrandtii (131)
t ^
and £. lanatus (26) has been identified as macrozamin. In view of the wide
occurrence of azoxyglycosides in various cycad plants, it is suggested that
all unprocessed cycad products should be considered potentially hazardous
until proven otherwise.
In addition to the rat, cycasin and cycad products are carcinogenic in
mice, hamsters, guinea pigs, rabbits, fish and monkeys. Cycasin is weakly
carcinogenic in adult C57BL/6 mice; a single intragastric dose of 300-1,000
mg/kg induced tumors (hepatoma, fibroma, lung and kidney adenoma) in only 4 of
35 mice (119). Like newborn rats, newborn mice are much more susceptible; a
single subcutaneous injection of 500 mg/kg cycasin was sufficient to induce
tumors in 100% of both dd (121) and C57BL/6 (120) mice. This susceptibility
decreases rapidly as the mouse ages. The tumor incidences of C57BL/6 mice
given a single s.c. dose of 500 mg/kg cycasin on day 1, 2, 4, 7 or 14 after
birth were 100, 100, 90, 73 and 15.7%, respectively (120). This age-
dependence may reflect a rapid postnatal decline in /3-glucosidase activity in
subcutaneous tissue of mice. Such a change has indeed been demonstrated by
Spatz (130) using rat skin homogenates.
244
-------
Besides purified cycasin, in a preliminary report by O'Gara et al. (122),
an aqueous extract of £. circinalis nuts was shown to induce liver and kidney
tumors in 3 of 11 C57BL mice following repeated topical applications to skin
ulcers artificially induced with 10% croton oil in mineral oil. This finding
is of special interest because of earlier reports on medicinal use of cycad
paste for the treatment of skin ulcers (1).
Regarding other animal species, Hirono et_ _al_. (123) showed that cycasin
is a weak to moderately active carcinogen in Syrian golden hamsters. Single
or repeated oral administration of 100-150 mg/kg cycasin to adult hamsters and
single subcutaneous injection of 200-600 mg/kg cycasin to newborn hamsters
induced mainly intrahepatic bile duct carcinomas: the incidence was in the
i ^
range of 12-17%. In addition, a few colon tumors, malignant lymphoma and an
occasional kidney or gall bladder tumor were observed. Spatz (124) maintained
guinea pigs on diets containing 5% cycad (C. circinalis L.) meal for several
5-day periods. Nine of 27 guinea pigs which survived for more than 44 weeks
developed liver tumors (4 hepatocellular carcinomas and 5 bile duct tumors).
Watanabe et al. (125) administered to each of 15 rabbits 1 ml of cycad (_£.
revoluta) extract (containing 16.6 mg cycasin) once a week for 27-33 weeks; 7
of 9 rabbits that survived over 200 days developed malignant liver tumors
identified as hemangioendotheliomas. White Leghorn chickens appear to be the
only animal species thus far reported to be refractory to the carcinogenic
effect of cycad products. Sanger et al. (126) found no tumors in chickens fed
0.5% cycad (C. circinalis) kernel for 68 weeks. In a preliminary communi-
cation, Stanton (34) reported that continuous feeding of a 50% dietary supple-
ment of cycad (_£. circinalis) nut meal to a species of aquarium fish
(Brachydanio rerie) led to the induction of liver foci, many showing the
histological characteristics of malignant neoplasms. The carcinogenic poten-
245
-------
tial of cycasin has also been investigated in nonhuman primates (rhesus,
cynomolgus and African green monkeys) by Sieber et al. (127). Eighteen
monkeys were given a combination of oral administration of crude cycad meal
for 10 months and purified cycasin for 3 years, followed by intraperitoneal
injections of synthetic MAM acetate for 33-92 months. At the time of the
report, 2 of 9 necropsied monkeys had developed hepatcellular carcinomas; one
of them also had an intrahepatic bile duct adenocarcinoma, a kidney carcinoma,
as well as adenomatous polyps of the colon. For comparison, only 4 of 143
historical controls developed tumors. In view of the proven carcinogenicity
of MAM in monkeys (see Section 5.3.2.2.3.2), it is not possible to assess from
this study (127) whether oral administration of cycasin alone is carcinogenic
, • "»
in monkeys.
5.3.2.2.3.2 CARCINOGENICITY OF METHYLAZOXYMETHANOL ACETATE AND RELATED
COMPOUNDS
The evidence that methylazoxymethanol (MAM), the aglycone of cycasin, is
the proximate carcinogen of the azoxyglycoside was first presented by
Matsumoto and Strong (24). An ethylether-soluble fraction of cycad (_£.
circinalis L.) nuts, when fed to three rats for a period of 9 months, at a
level equivalent to 2.5% cycad nut in the feed, induced hepatomas. The frac-
tion was found to contain free MAM. Laqueur and Matsumoto (107, 132) provided
further evidence that, unlike cycasin, MAM is carcinogenic when injected into
rats. The carcinogenicity target organs of MAM are the same as those of
cycasin (see Table XLII). Both conventional and germ-free rats are suscep-
tible to the carcinogenic effect of MAM (111). Free MAM is also carcinogenic
in Syrian golden hamsters and in guinea pigs (13). Of 64 hamsters that
received a single or multiple (up to 5) i.p. or i.v. injection(s) of 10-20
mg/kg MAM, forty developed adenocarcinomas of the large intestine, nine had
246
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p. 1 of 2
Table XLII
Carcinogenicity of Free Methylazoxymethanol (MAM), Synthetic MAM Acetate
and Related Compounds
Species and Strain9
Route
Princ ipal
Organs Affected
References
(A) Free Methylazoxyraethanol (MAM)
Rat , —
Rat , Fischer
Rat , Sprague-Dawley
(conventional or GF)
Hamster, Syrian golden
Guinea pig
(B) Methylazoxymethanol
Rat, Sprague-Dawley
(GF)
Rat, Fischer 344
Rat, Sprague-Dawley
Rat , Sprague-Dawley
(GF)
Rat, Wistar (newborn)
Rat, Buffalo
Rat, Sprague-Dawley
(GF)
Rat, Sprague-Dawley
or Lobund Wistar
Rat , Donryu
oral
i.p.
i.p.
i.p. or i .v
i.p.
Acetate (MAM
oral
oral
i .v . or i.p
i.p.
i.p.
i.p.
s .c .
s .c.
intrarectal
Liver
Intestines ,
kidney, liver
Intest ines
Colon, liver
Liver, nasal
cavity
Acetate)
Kidney, liver,
intest ines
Colon, stomach,
kidney, Zymbal's
gland, liver
Intestines,
kidney, liver
Intestines ,
liver, kidney
Kidney
Colon
Intestines ,
liver
Intestines ,
liver
Large intes-
(24)
(107, 132_)
(111)
(13)
(13)
(111)
(133)
(35, 134, 135)
(111)
(118)
(136)
(111)
(137)
(138)
tine, kidney,
liver
-------
Table XLII (continued)
p. 2 of 2
Species and Strain
Mouse, CD-I
Mouse, SWR/J or
C57BL/6J
Mouse, AKR/J
Mouse, BALB/c
Guinea pig
Medaka (Oryzias
lat ipes)
Monkey ( rhesus ,
cynomolgus or
African green)
(C) Methylazoxymethanol
Rat, Wistar (weanling)
Rat, Wistar (newborn)
Rat, Wistar
(D) Methylazoxymethanol-
Rat , Sprague-Dawley
Rat , Sprague-Dawley
(GF)
Principal
Route Organs Affected
i.v. None
s.c. Colon, rectum,
anus
s.c. Hematopoiet ic
system
Perianal Anus, vascular
painting system
i.p. or s.c. Liver, vascular
system
oral Liver
i.p. Liver, kidney,
esophagus ,
intest ines
Benzoate (MAM Benzoate)
oral Intestines
s.c. Kidney, colon
s .c . Kidney, 1 iver
6-D-glucosiduronic acid (MAM glucuron
oral Colon, small
intestine ,
liver, kidney
i.p. Small intes-
tine (marginal
act ivity)
oral or i.p. None
References
(35, 134)
(139)
(139)
(140, 141)
(13)
(142)
(127)
(118)
(118)
(118)
ide)
(143)
(143)
(143)
*GF = germ-free
-------
liver tumors, and five had gall bladder or intraorbital carcinomas. Among the
20 guinea pigs given a total dose of 9-19.5 rag MAM intraperitoneally, 14
developed tumors which included 10 liver tumors, 4 squamous cell carcinomas
or hemangiosarcomas of the nasal cavity, 2 lung adenomas and 1 jejunal adeno-
carcinoma. In both experiments, none of the control hamsters or guinea pigs
developed tumors.
The role of MAM as the proximate carcinogen of cycasin and related azoxy-
glycosides, as well as of 1,2-dimethylhydrazine and azoxyalkanes (see Section
5.2.1.3.4, Vol. IIIA), and its possible usefulness as a model experimental
carcinogen have created considerable interest for the study of the compound.
However, because of the instability of free MAM, most of the studies have been
carried out with the much more stable synthetic compound, MAM acetate (see
Table XLII). The synthetic MAM acetate has been shown to be carcinogenic in 6
strains of rats via 5 different routes of administration inducing tumors
mainly in the large intestine, kidney and liver. Some minor strain and sex
differences have been noted. Laqueur et al. (Ill) treated germ-free Sprague-
Dawley rats with MAM acetate (total dose 12.5-13.7 mg administered over a
period of 14 to 21 days) via oral, i.p., and s.c. routes. The i.p. route was
the most effective, inducing colon carcinomas in 4 of 4 rats and hepatic bile
duct adenomas in 2 of 4 rats. Rats that received MAM acetate in the diet
developed more kidney tumors and less intestinal tumors than those treated
parenterally. McConnell £t__al_. (133) conducted a dose-response relationship
study of MAM acetate in Fischer 344 rats of both sexes. The animals were
given 5 doses each of 0, 0.2, 1.0, 4.0, 7.5, 15 or 30 mg/kg MAM acetate via
gavage at 2-week intervals for 14 months; the resulting tumor incidences were
1.7, 1.9, 0, 3.7, 8.6, 37.5 and 75%, respectively. The male rats developed
more colon tumors at lower doses than the female rats. Tumors of Zymbal's
247
-------
gland and of the kidneys were found exclusively in male and female rats,
respectively. Zedeck et al. (35, 134, 135) administered a single i.v. or i.p.
dose of 35 mg/kg MAM acetate to Sprague-Dawley rats and induced tumors in
13/20 and 29/33 rats, respectively. Most of the tumors developed in the large
intestine; however, some were found in the small intestine, kidney and
liver. Hayashi et al. (118) found the kidney to be virtually the only target
organ in Wistar rats that received a single i.p. dose of 20-30 mg/kg MAM
acetate within 24 hours -after birth. In Buffalo-strain rats, however, the
descending colon was the predominant carcinogenicity target organ of repeated
i.p. injections of 20 mg/kg MAM acetate (136). In a comparative study by
Pollard and Zedeck (137), all animals in groups of Sprague-Dawley and Lobund
i- ^
Wistar rats which received 10 weekly s.c. doses of 30 mg/kg MAM acetate
developed colon tumors. However, the Sprague-Dawley rats were much more
susceptible than the Lobund Wistar rats as indicated by the substantially
greater tumor multiplicity (20.4 vs. 3.1). Narisawa and Nakano (138) infused
1 mg MAM acetate into the rectum of Donryu rats once a day for 7-21 days.
Fifty-four weeks after the initiation of carcinogen administration, 22 of 24
treated rats developed multiple carcinomas in the large intestine. Seven rats
also had nephroblastomas and two had hepatocellular adenomas.
Much greater strain differences in the response of mice to MAM acetate
administration have been noted. Zedeck _et_ al_. (35, 134) found no tumors in
CD-I mice following injection of a single i.v. dose of 35 mg/kg of the com-
pound. On the other hand, Diwan £t_ _al_. (139) induced tumors in 20/21 SWR/J,
21/26 C57BL/6J and 22/28 AKR/J mice by injection of 10 s.c. doses of 20 mg/kg
MAM acetate; the corresponding incidences in control mice were 2/20, 0/22 and
17/24, respectively. Most of the tumors in the SWR/J and C57BL/6J mice were
in the large intestine. In contrast, AKR/J mice were resistant to the colo-
248
-------
rectal c.rcinogenic effect of MAM acetate; the only type of tumor detected was
leukemia, which is known to occur spontaneously in this strain. In BALB/c
mice, topical painting of 0.5 mg MAM acetate at the anal region, three times
weekly for 24 weeks, led to the induction of tumors of the perianal sebaceous
gland in 23 of 24 (96%) male and 16 of 30 (59%) female mice. Vascular tumors
of the liver and fat tissue of the abdominal cavity also developed in 16 of 24
male and 3 of 30 female mice. It was suggested that anatomical difference in
the external genitalia in the two sexes contributed to sex differences
described above.
Among other animal species tested, guinea pigs, raedaka and monkeys are
all highly susceptible to tne carcinogenic effects of MAM acetate. Laqueur
and Spatz (13) reported that in a group of 25 guinea pigs, given repeated s.c.
or i.p. injections of MAM acetate (total dose 44-77 mg), all developed hepato-
cellular carcinomas, and 14 of them also had vascular tumors. None of the 59
controls bore tumors. In the study of Aoki and Matsudaira (142), who intro-
duced MAM acetate into the aquarium water of medaka (Oryzias latipes) at
levels of 0.1-3 ppm for periods ranging from 1 to 120 days, over 80% of the
surviving fish developed liver tumors 2-5 months after the commencement of the
treatment. No tumors were found in 74 control fish within the same period.
Sieber et al . (127) dosed 10 old world monkeys (rhesus, cynomolgus, and
African green) with weekly i.p. injections of 3-10 mg/kg MAM acetate for life
beginning within 72 hours after birth. Six of the 8 monkeys, that died after
45-89 months of treatment, developed tumors which were identified as 5 hepato-
cellular carcinomas, 3 esophageal squamous cell carcinomas, 2 renal carci-
nomas, and 1 multifocal adenocarcinoma of the small intestine. Liver biopsy
of one of the two remaining survivors revealed a tumor mass identified as a
well-differentiated hepatocellular carcinoma.
249
-------
Two other synthetic derivatives of MAM have been tested for carcinogenic
activity (see Table XLII). Hayashi et al. (118) induced tumors in 7 (6
kidney, 1 colon) of 13 Wistar rats that survived a single s.c. dose of 40-60
mg/kg MAM benzoate. By oral route (4 x 5 mg via stomach tube), MAM benzoate
induced tumors in both the small and the large intestine of 5 of 6 treated
rats. Subcutaneous injections (2x5 mg) of the compound to young adult rats
led to the development of one hepatoma and one nephroblastoma in the 2 surviv-
ing animals.
In an effort to test the hypothesis that biliary excretion may play a
role in the intestinal carcinogenic action of cycasin and its aglycone (see
144), MAM-A-D-glucosiduronic>acid was synthesized and bioassayed (143).
Conventional and germ-free Sprague-Dawley rats were given a single or 4 weekly
i.p. or oral doses of 70.5 mg/kg of the compound. Twenty-seven of the 30
orally treated conventional rats developed tumors, which were predominantly in
the large intestine and occasionally in the liver and kidney. Among the i.p.
dosed conventional rats, only two adenomas were found in the small intes-
tine. The MAM glucuronide was completely inactive in germ-free rats by either
oral or i.p. route. The study did not support the biliary excretion hypo-
thesis. Mammalian p-glucuronidase was apparently unable to hydrolyze MAM-p -
D-glucosiduronic acid to release MAM.
5.3.2.2.3.3 TRANSPLACENTAL CARCINOGENESIS BY CYCASIN AND ITS AGLYCONE
The transplacental carcinogenic action of cycasin was first shown by
Spatz and Laqueur (145). Pregnant Sprague-Dawley rats were fed diets supple-
mented with 1, 3 or 5% cycad meal (which contained 3% cycasin) during the 1st,
2nd or 3rd week of gestation or throughout the gestation. Of the 81 offspring
that survived more than 6 months, 15 (18.5%) had tumors. These included
250
-------
mesenchymal tumors in the jejunum in 4 rats from the same litter (exposed in
utero during days 2-5 of gestation), brain tumors (gliomas) in 5 rats (from 5
different litters) and a variety of other tumors (mostly exposed during days
14-18 of gestation). To determine the influence of the period of fetal
development at the time of carcinogen exposure on the location and types of
tumors, Laqueur and Spatz (13, 146) gave pregnant Fischer rats a single i.p.
or i.v. dose of 20 mg/kg MAM or MAM acetate at various days of gestation.
Among 340 offspring examined, 42 (12.3%) had tumors. Most of the tumors
developed in the lungs, brain, kidneys and intestines. As many as 19 of these
42 tumor-bearing animals were among the progeny of 9 mothers who had received
the carcinogen on the 21st jda^y of pregnancy. These rats accounted for 10 of
the 16 pulmonary tumors and 6 of the 7 gliomas found. Thus, the last day of
gestation was not only the most susceptible period but also reflected a
heightened sensitivity of the pulmonary and cerebral tissues to the carcino-
genic action of MAM. In a more recent study by Kalter _et _al_. (101), MAM
acetate caused microencephaly (see Section 5.3.2.2.2.2) but produced no neuro-
genic tumors in Sprague-Dawley rats, when injected intraperitoneally at a
single dose of 20 or 30 mg/kg on the 15th day of gestation. When administered
together with N-ethyl-N-nitrosourea (ENU; a potent transplacental carcinogen,
see Section 5.2.1.2.3.6), MAM acetate inhibited rather than potentiated the
transplacental carcinogenic action of ENU. The site of tumor inhibition by
MAM acetate was confined to the brain, coinciding with the site of teratogenic
damage by the compound. The inhibitory effect of MAM acetate was attributed
partly to the destruction of ENU-sensitive embryonic brain cells and partly to
other mechanisms, such as blocking of molecular target sites denying access to
ENU (101).
251
-------
The evidence that cycasin and its aglycone, MAM, can indeed cross the
placental barrier was demonstrated by Spatz and Laqueur (147). Both cycasin
and MAM were detected in 15-day-old rat fetuses 3-24 hours after a single oral
administration of cycasin to pregnant rats. Cycasin and MAM were also found
in the maternal mammary glands and in suckling rats indicating that newborn
animals can be exposed to cycasin through lactation. This finding is of
particular importance because newborn rats (and possibly other newborn
animals) contain /3-glucosidase (130) and are, thus, highly susceptible to the
carcinogenic action of cycasin. Up to 20% of the MAM can be recovered from
hamster fetuses 10-30 minutes after a single i.v. injection of 2.5-20 rag MAM
to pregnant hamsters on day 11 of gestation. Owing to the high chemical
reactivity of the compound, only a trace was found after 3 hours and none
after 4 hours (147). Using 3H-labeled MAM acetate, Nagata and Matsumoto (148)
showed that MAM crossed the placenta in rats and became covalently bound to
fetal DNA, RNA and proteins.
5.3.2.2.3.4 MODIFICATION OF CARCINOGENESIS BY CYCASIN AND MAM ACETATE
Cycasin acts synergistically with several chemical carcinogens and
tumorigenesis promotors. Mori and Hirono (149) fed Sprague-Dawley rats a
coffee solution (equivalent to the amount normally consumed by humans) for 480
days and/or administered a single intragastric dose of 150 mg/kg cycasin on
the 121st day of the experiment. Twelve of 35 rats that received both coffee
and cycasin developed tumors at various sites with the colon and the rectum
being the most affected sites. For comparison, only one of 18 rats treated
with cycasin alone had a kidney tumor and no tumors were found in 18 rats
given coffee alone. The investigators attributed the potentiating activity of
the coffee solution to its cocarcinogenic constituents (e.g., chlorogenic
acid). Uchida and Hirono (150) showed that chronic administration of pheno-
252
-------
barbital (0.05% in diet) enhances the incidence of liver tumor (from 9% to
63.6%) in female ACI rats given a relatively low (marginally carcinogenic)
dose of 100 mg/kg cycasin. The tumor incidences at other sites, or in male
rats, were not significantly affected and phenobarbital alone was not carcino-
genic. This finding gives further support to the body of evidence showing
that phenobarbital is an effective promoter of hepatocarcinogenesis (see
Section 5.2.1.7.11 in Vol. IIIA) . Davis £t_^. (151) demonstrated that
cycasin (160 ppm in cycad nut meal) acted synergistically with dipentylnitros-
amine (1,500 or 2,000 ppm in diet) in inducing liver tumors in Fischer 344
rats. Davis and coworkers hypothesized that dipentylnitrosamine-induced
hyperplastic nodules can resist the cytotoxic effect of cycasin and progress
• >
rapidly toward hepatomas while other areas of the liver are cytotoxically
affected.
Methylazoxymethanol (MAM) acetate has been extensively used as a model
chemical carcinogen in the study of various modifying factors and in the
development of new chemotherapeutic agents. As may be anticipated, two anti-
oxidants, butylated hydroxyanisole (152, 153) and selenium (154, 155), inhibit
the carcinogenicity of MAM acetate toward the colon of rats and mice, while
high-fat diet (156), marginally lipotrope-deficient diet (157), and surfac-
tants (158) have the opposite effect. Colostomy (establishment of an artifi-
cial anus by an opening into the proximal end of the colon) had no significant
effect of MAM acetate-induced carcinogenesis in the distal end of the colon of
rats indicating that the carcinogen can reach the intestinal mucosa via the
vascular system as well as via the fecal stream (159). Partial hepatectomy 24
hours prior to the administration of MAM acetate increases only slightly the
incidence of liver tumors, suggesting that both dividing and resting liver
cells are susceptible to MAM acetate carcinogenesis (160). Chronic admini-
253
-------
stration of hydrogen peroxide (1.5% in the drinking water) before and after a
single intraperitoneal injection of MAM acetate substantially increases the
incidence of tumor in duodenum (161); the mechanism of this unusual potentia-
tion remains to be studied. Pyrazole, an inhibitor of alcohol dehydrogenase,
significantly inhibits MAM acetate-induced colon/intestine carcinogenesis
(162, 163) in rats, implicating the involvement of the enzyme in the activa-
tion of the carcinogen. In contrast to the colon, pyrazole administration
enhances MAM acetate-induced kidney and skin carcinogenesis (163). It has
been suggested that rat kidneys contain a choline dehydrogenase which can
activate MAM acetate and is not inhibited by pyrazole; furthermore, inhibition
of liver and colon alcohol dehydrogenase by pyrazole causes a shunting of MAM
acetate toward the kidneys and other organs (163, 164). Methylazoxymethanol
acetate-induced colon carcinogenesis has been used as a model system for
testing chetnotherapeutic agents. Drugs which have been shown to be effective
in inhibiting this tumor growth or causing tumor regression include indo-
methacin (165, 166), hydrocortisone (165), 4'-deoxyrubicin (167), 5-fluoro-
uracil (167) and piroxicam (168). Dietary restriction also inhibits MAM
acetate-induced colon carcinogenesis; however, it is effective only if it is
implemented immediately after administration of the carcinogen (169).
5.3.2.2.4 Metabolism and Mechanism of Action
The metabolism and mechanism of action of cycasin and its aglycone,
methylazoxymethanol (MAM) have been extensively studied. The known metabolic
pathways are outlined in Fig. 10. Several lines of evidence concur that
enzymatic deglucosylation of cycasin to MAM is the first and obligatory step
in the metabolic activation of cycasin to genotoxic and toxic interme-
diated). Cycasin is toxic (31) and carcinogenic (9, 106, 107) in adult rats
after oral administration but is apparently innocuous by intraperitoneal
254
-------
fl-qlucosidase
Cycasin
glucose
0
t
CH3-N=N-CH2OH
(MAM)
NAD+
NADH
0
* //
CH3-N=N-C
0
t
CH3-N=N-CH2OAc
(MAM acetate)
slow
(MAMAL)
u
M
HCHO
HCOOH
[CH3-N=N-H]
r -,
rcH3-N=N-om
r® -i
[CH3]
Fig. 10. Proposed metabolic pathways of cycasin and methylazoxymethanol
acetate. Abbreviations: MAM, methylazoxymethanol; ADH, alcohol dehydrogenase;
MAMAL, methylazoxymethanal.
-------
injection. This dramatic difference in activity appears to be due to metabo-
lism in the intestine, because almost 100% of the parenterally administered
cycasin is excreted unchanged in the urine of rats, while only 30-60% is
excreted by rats which received cycasin by intragastric administration (25).
This is supported by the finding that germ-free rats, which are resistant to
the toxicity and carcinogenicity of cycasin (106, 111), also fail to metabo-
lize ingested cycasin (128). Establishing an intestinal bacterial flora in
germ-free rats by oral administration with Streptococus fecalis (which has
f3-glucosidase activity) confers to the rats the ability to metabolize cycasin
and susceptibility to the toxicity of the compound (129). Both conventional
and germ-free rats are susceptible to the toxicity and carcinogenicity of MAM,
the aglycone of cycasin, irrespective of the route of administration (see
Section 5.3.2.2.3.2).
Studies by Hirono and associates (33, 119-121, 123) demonstrated that,
unlike adults, newborn animals are susceptible to carcinogenicity of parenter-
ally administered cycasin. Susceptibility rapidly decreases as the animal
ages. Laqueur and Spatz (13, 146) found that rats are most sensitive to the
transplacental carcinogenic action of cycasin when administered one day before
birth. These observations can be best explained by the ontogenic development
of the mammalian p-glucosidase. Spatz (130) detected -glucosidase activity
in the skin (as homogenates) of fetal and neonatal rats from 15 days before
birth to 30 days after birth; activity peaked around the time of birth (2 days
prenatal to 6 days postnatal) and declined sharply afterwards. Matsumoto et
al. (27) found that the ^-glucosidase activity in the small intestine of
preweanling rats reaches a maximum on the 15th postnatal day and decreases
steadily thereafter.
255
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Further support for the crucial role of enzymatic deglucosylation in the
metabolic activation of cycasin is provided by various mutagenicity studies
(&ee Section 5.3.2.2.2.2). Cycasin is nonmutagenic in virtually all in vitro
studies using bacteria, yeasts or cultured mammalian cells, but is activated
by p-glucosidase, fecalase or whole intestinal bacteria flora. In host-
mediated assays, cycasin is mutagenic only by oral administration to the host;
activity is abolished by pretreating the host with an antibiotic which wipes
out or drastically curtails the intestinal flora. Among higher plants and
insects, cycasin displays mutagenic or clastogenic activity only in cells or
organisms which contain /3-glucosidase activity. On the other hand, MAM or
MAM acetate is mutagenic in ,a>wide variety of test systems often without
further metabolism.
Methylazoxymethanol is a relatively unstable compound which readily
decomposes to yield formaldehyde, nitrogen and methanol (see Section
5.3.2.2.2.1). Under physiological conditions, MAM spontaneously hydrolyzes in
aqueous solution with a half-life of about 11.5 hours (28). The chemically
more stable synthetic derivative, MAM acetate, is expected to be readily
converted to MAM after in vivo deacetylation by esterases. For example, serum
cholinesterases have been shown to hydrolyze MAM acetate (170). Free MAM is a
good alkylating agent upon decomposition. _p_-Chlorobenzoic acid, acetic acid,
phenol and the guanine moiety of DNA and RNA are methylated by incubation with
MAM at 37°C for several hours (28, 171). The biochemical action of MAM is
strikingly similar to that of diraethylnitrosatnine (172). Hydroxyraethylmethyl-
nitrosamine, the unstable, raetabolically activated intermediate of the
nitrosamine (see Vol. IIIA, p. 253) and MAM are isomers and it has been sug-
gested (173) that the two compounds produce the same transient reactive inter-
mediates. As in the case of dimethylnitrosamine, the idea that diazomethane
256
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may be a possible reactive decomposition product of MAM has been discarded,
because hydrolysis of MAM in the presence of D20 does not yield the antici-
pated deuterated methanol (174). Methylcarbonium ion and methyldiazonium
hydroxide are considered to be the probable methylating intermediates.
Investigations by Zedeck and associates indicate that further metabolism
of MAM (see Fig. 10) to its aldehydic form, methylazoxyformaldehyde or methyl-
azoxymethanal (MAMAL) may play an important role in the activation of the
compound, in addition to the products generated by the spontaneous decomposi-
tion of MAM. Following the suggestion by Schoental (175) that MAM may be
activated through oxidation to MAMAL by alcohol dehydrogenase (ADH), Zedeck
and associates (28, 162, 176', 177) have demonstrated that MAM can indeed serve
as a substrate for this cytosolic enzyme. The reaction requires NAD or NADP
as a cofactor. Tissues which are sensitive to the toxic and carcinogenic
effects of MAM (e.g., liver, colon, cecum) contain high levels of
NAD -dependent ADH activity, whereas those which are relatively resistant to
MAM (e.g., jejunum, ileum) contain little ADH activity. Furthermore, treat-
ment of rats with pyrazole, an inhibitor of ADH, protects the animals against
the toxicity (162, 176) arid colonic carcinogenicity (155, 162, 163) of MAM.
However, pyrazole treatment was found to enhance rather than to inhibit the
renal and skin carcinogenicity of MAM (163). This apparent paradoxical effect
can be partly explained by the finding of Tan et al. (164) that kidneys (and
liver) contain another dehydrogenase (choline dehydrogenase) that oxidizes MAM
and is not inhibited by pyrazole. The inhibition of ADH in the colon and
liver by pyrazole is also expected to make more MAM available to act upon the
kidneys and other organs. The exact role of MAMAL as a reactive intermediate
of MAM and cycasin is not clearly understood. Feinberg and Zedeck (28)
reported that MAMAL spontaneously decomposes to yield methylating inter-
257
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mediates at a substantially faster rate than does MAM. Thus, MAMAL is likely
to be a better alkylating agent than MAM. Furthermore, it has been suggested
that MAMAL may act as a cross-linking agent (175) or directly react with the
amino group of macromolecules to form a Schiff base or an acylated (N-formyl)
derivative (28); however, evidence for such adduct formation is still lacking.
Biliary excretion of glucuronidated MAM was at one time hypothesized to
account for the predilection of MAM for inducing intestinal tumors. This
£>
hypothesis has been tested by several groups of investigators, using different
approaches yielding consistently unsupportive results. Matsumoto (144)
injected synthetic glucuronide of MAM (MAM-$-D-glucosiduronic acid) into rats
and was unable to recover the* compound in the bile. Rats injected with free
MAM also did not excrete the glucuronide in the bile. Cannulation of the bile
ducts of rats had no effect on MAM-induced inhibition of DNA synthesis in the
epithelium of various segments of the intestines (177). Yet, this site-
specific biochemical event was well correlated with the eventual emergence of
tumors. Matsubara et al. (159) showed that colostomy (see Section
5.3.2.2.3.4) had no significant effect on MAM acetate-induced colon carcino-
genesis, indicating that the carcinogen can reach colon via vascular system as
well as via fecal stream.
The mechanism of carcinogenic action of cycasin and its aglycone is not
clearly understood but is generally believed to involve covalent binding of
alkylating intermediate(s) to cellular macromolecules as the first step.
7-Methylguanine has been isolated from the hydrolysates of both DNA and RNA
after incubation with MAM at 37°C for 16 hours (171). Like several other
colon carcinogens, MAM acetate binds to cellular DNA and protein in explant
cultures of rat colon (178). In vivo methylation of nucleic acids in several
other target organs of cycasin and MAM — the liver and kidney of adult rats
(172) and the brain of rat fetuses (148) — have also been demonstrated.
258
-------
The early biochemical and cell morphological effects of MAM have been
extensively studied. Inasmuch as a single administration of cycasin or MAM
acetate can result in tumor induction, events occurring immediately after
exposure may be of crucial importance to the initiation of carcinogenesis.
Zedeck and associates (134) reported that MAM inhibited DNA synthesis in the
three principal target organs (liver, kidney, intestine) of the carcinogen in
rats within a few hours after treatment. No such activity was detected in
CD-I mice which are resistant to the carcinogenic effect of MAM acetate.
Nucleolar structural alterations (occurring within 15 minutes after treatment
and persisting for months thereafter), inhibition of RNA (both nuclear and
nucleolar) and protein synthesis have also been noted in rat liver (35).
Mitotic abnormalities were evident in hepatic cells 7 days after treatment
when the rate of DNA synthesis had already returned to normal level. The
number of polyploid cells increased significantly and remained elevated for as
much as one month after treatment (95). Various segments of rat intestine
displayed differences in acute response (inhibition of DNA synthesis and
mitosis) to MAM acetate treatment. The segments which are the most acutely
affected (e.g., colon, cecum, duodenum) are also the sites of eventual tumor
emergence (177). It is of interest to note that MAM acetate causes inhibition
of DNA synthesis in human colon mucosa in organ culture (179), suggesting that
the human colon may be susceptible to the carcinogen.
The mechanism of inhibition of macromolecular synthesis by MAM has been
investigated. Grab _et_ al. (180) found no significant change in the template
capacity of hepatic DNA chromatin from MAM-treated animals but noted conforma-
tional changes in some "aggregate" enzyme preparations. They suggested that
the induction of such changes in hepatic nuclear protein may result in
decreased RNA synthesis. The studies of Yu et al. (181), however, indicated
259
-------
that MAM acetate inhibits RNA synthesis by both impairing the chromatin tem-
plate function and selectively inhibiting RNA polymerase II. The impairment
of template function may be a direct result of DNA methylation or a conse-
quence of MAM acetate-induced chromatin condensation. The selective inhibi-
tion of RNA polymerase II appears to result from direct modification of exist-
ing enzyme to a catalytically deficient state. The possible mechanism of
hepatic protein synthesis by MAM acetate has also been studied (182). No
significant alterations of ribosomal subunits and of initiation factors were
found suggesting that the inhibition may result from an alteration of cyto-
plasmic mRNA and its association with ribosomes.
5.3.2.2.5 Environmental Significance
The environmental occurrence of cycasin and related compounds, the human
uses of cycad plants, and their potential health hazards have been discussed
in a variety of review articles (1, 15-18, 144). Cycasin, macrozamin and
other azoxyglycosides occur in the seeds, stems, roots and leaves of cycad
plants which are indigenous in the tropical and subtropical regions around the
world and occasionally in temperate zones such as in Florida, Japan and
Australia. Among the 9-10 genera of cycads identified, the most widely
distributed genus is Cycas. The growth zone of this variety extends from East
Africa and Madagascar across the Indian Ocean to the Mariana Islands (Guam)
and Japan. Zamia plants are located in Florida, the Carribean Islands, Mexico
and the northern parts of South America. Macrozamia and Bowenia are the two
most commonly encountered genera in Australia whereas Encephalartos and
Stangeria are found in East, Central and South Africa.
The amount and the type of azoxyglycosides found in cycad products depend
on the species of cycad, the condition of the products, and the method of
260
-------
extraction. Cycasin occurs in two important Cycas species — C^. circ inalis L.
(in Guam) and C_. revoluta Thunb (in Japan); its occurrence in Zamia plants has
also been suggested (110). The reported yield ranged from 0.02 to 5% (24,
183, 184). In general, fresh and unwashed cycad nuts contain substantially
higher amounts of cycasin than washed, fermented and dried cycad nuts. Boil-
ing during the extraction procedure improves the yield by destroying the
cycasin-destroying enzyme (emulsin) present in the cycad extract. Samples of
dried cycad chips, prepared by Guamanians for human consumption, contained no
detectable amount of cycasin (183). Small amounts of transglycosylated deri-
vatives of cycasin (neocycasin A, B, C and E) have also been isolated from C.
revoluta Thunb (20-23). Macspzamin was first obtained from Macrozamia
spiralis (2) and has since been found in several different species of
Microzamia (3), in Encephalartos species (26) , and in Zamia leaves (110) . The
reported yield from one of these studies was 0.1% (26).
Various parts of cycad plants are still used as a source of food and
medicine by local inhabitants in some parts of the world (1, 16-18). A major
food use of cycads is as cycad flour or starch. After elaborate washing and
fermentation procedures the final products appear to be free of cycasin. No
cycasin was detected in samples of Guamanian homemade cycad (C_. circinalis L.)
flour (108) and dried cycad chips (183), and in Japanese homemade cycad bean
paste "sotetsu miso" (185). Between 1845 and 1920, cycad starch was produced
commercially in Florida using Z. floridana (1). Incidents of human poisoning
have been reported (1, 51) during times of food shortage and famine when
inadequately detoxified cycad products were consumed. In addition to their
use as flour or starch, fresh cycad husk is eaten as candy by the natives of
Guam. Seeds, gum, stems, leaves or roots of Cycas and Zamia plants were used
medicinally to treat snake or insect bites, as emetic, laxative, aphrodiziac
261
-------
and for various other purposes in India, China, Mexico and southeast Asia
(1). Roots or barks of Encephalartos and Stangeria spp. were also used medi-
cinally by African natives (117).
Despite the proven animal carcinogenicity of cycasin and various cycad
products, there is no epidemiologic evidence in support of their human car-
cinogenicity. Mugera and Nderito (114) suggested that cycads (Encephalartos
and Stangeria^ spp.) may play a role in the etiology of liver cancer among East
African natives but provided no supportive documentary evidence. African
natives are known to be exposed to other liver carcinogens such as aflatoxins
(see Section 5.3.1.1.5.1). Between 1961 and 1965, Hirono ^t__al_. (51) carried
out an epidemiologic study of* the inhabitants of Miyako Islands, Okinawa.
These natives were forced by circumstances to subsist on cycads (_C_. circinalis
L.) during a period of food shortage, after a series of typhoons had struck
the islands in 1959. An unusually high incidence of liver cirrhosis was
observed among the natives. However, no significant increase in cancer mor-
tality was found. It has been pointed out (17) that the results may be nega-
tive because of the short follow up time or because the cycad foods were well
prepared. Judging from the absence of cycasin in well prepared cycad foods,
it appears that the carcinogenic risk of consumption of cycad foods is low.
However, human consumption of unprocessed cycad material should be totally
avoided.
262
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98. Spatz, M. , and Laqueur, G.L.: Proc. Soc. Exp. Bipl. Med. 129, 705
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101. Kalter, H., Ormsby, I., and Warkany, J.: Biol. Res. Pregnancy 3, 93
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106. Laqueur, G.L.: Fed. Proc. 23, 1386 (1964).
107. Laqueur, G.L.: Virchow's Arch. Path. Anat. 340, 151 (1965).
108. Yang, M.G., Mickelsen, 0., Campbell, M.E., Laqueur, G.L., and
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109. Yang, M.G., Sanger, V.L., Mickelsen, 0., and Laqueur, G.L.: Proc. Soc.
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110. Hoch-Ligeti, C., Stutzman, E., and Arvin, J.M.: J. Natl. Cancer Inst.
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111. Laqueur, G.L., McDaniel, E.G., and Matsumoto, H. : J. Natl. Cancer
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112. Fukunishi, R. , Watanabe, K., Terashi, S., and Kawaji, K.: Gann 62, 353
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113. Gusek, W., and Mestwerdt , W. : Beitr. Path. Anat. 139. 199 (1969).
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136. Reuber, M.D.: Digestion 14, 311 (1976).
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160. Zedeck, M.S., and Sternberg, S.S.: Chem.-Biol. Interact. 17, 291
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161. Hirota, N. , and Yokoyama, T.: Gann 72, 811 (1981).
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SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.3.2.2
1. Whiting, M.G.: Econ. Bot. 17, 271-302 (1963).
2. Laqueur, G.L., and Spatz, M.: GANN Monogr. Cancer Res. 17, 189-204
(1975).
3. Magee, P.N., Montensano, R. , and Preussmann, R.: N-Nitroso Compounds
and Related Carcinogen^ . In "Chemical Carcinogens" (C.E. Searle, ed.),
ACSMonog. No. 173, American Chemical Society, Washington, D.C., 1976,
pp. 491-625.
4. IARC: Cycasin. In "Some Naturally Occurring Substances," IARC Mono-
graphs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol.
10, International Agency for Research on Cancer. Lyon, France, 1976,
pp. 121-138.
5. Laqueur, G.L.: Oncogenicity of Cycads and its Implications. In
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6. Hirono, I.: CRC Grit. Rev. Toxicol. 8, 235-277 (1981).
7. Morgan, R.W., and Hoffmann, G.R.: Mutat . Res. 114. 19-58 (1983).
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275
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