BEOLOGECAL EFFECTS OF m; ENGESTION
0r THE Tom PLANT,
CYCAS CERCINALI§

 

Thesis gov HM Dogma of M. S.
MCHEGRN STATE UNIVERSETY
Margaret Elizabeth Campbeil
1964

 

LIBRARY

Michigan State
University

 

%‘—_--.._

ABSTRACT

BIOLOGICAL EFFECTS OF THE INGESTION OF THE
TOXIC PLANT, CYCAS CIRCINALIS

 

by Margaret Elizabeth Campbell

Weanling albino rats and several species of farm
animals were used to study the toxic effects of the consump-
tion of Cycas circinalis. Parts of this plant, particularly
the flour prepared from the nut are of value as human food.

Cycad materials were incorporated on a percentage
basis into natural grain rations of the experimental animals.
Flour prepared from the fresh nut and from the nut washed
for the purpose of destroying the toxic prOperties, were
studied. Cycasin, the toxic glucoside isolated from the nut
and believed responsible for the toxicity thereof was also
used in feeding trials. The outer husks of the nut were fed
in a third series of studies.

A total of 485 weanling albino rats were used in 11
short term bioassays. These were designed to study vari-
ations in the animals response to the toxicity, the sta-
bility of the toxic factor under various conditions and the
effects of dietary change on the biological response of the
animals. Body weight changes and pathological lesions were

used to evaluate the relative toxicity of the cycad materials

Margaret Elizabeth Campbell

used. Data collected show these factors exerted an influence
on the biologic response.

Long term trials were conducted in which the response
of rats fed diets containing 1 or 1.5% cycad were compared
with that of rats fed 200 or 400 ppm cycasin. A trial last-
ing 23 months was designed to study the nutritional safety
of washed cycad flour.

Studies in which pigs, rats and cattle were used
demonstrated that the toxic factor in cycad flour passes
through the placenta and mammary gland. Additional bioassays
showed that dimethylaminoazobenzene, diethylnitrosamine and
dimethylnitrosamine behaved similarly to the toxic compound
in unwashed cycad.

Attempts were made to identify that fraction of cow's
milk that contained the toxic factor of cycad. The results
of two trials in which rats were fed milk fractions from a
cycad-fed-cow were not conclusive.

The husks of the cycad nut caused a toxic response
that appears to be different from that caused by cycad nut
flour. Hemoglobin and hematocrit values indicated the early
development of hemolysis and vasoconstriction which.‘were not
apparent in animals fed cycad flour.

Cattle, horses and pigs were all susceptible to the
toxicity of the nut flour. Their responses were similar to

those seen in rat feeding trials.

BIOLOGICAL EFFECTS OF THE INGESTION OF THE

TOXIC PLANT, CYCAS CIRCINALIS

 

BY

Margaret Elizabeth Campbell

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition

1964

ACKNOWLEDGMENTS

For his help, guidance and unending kindness, I ex-
press my sincere gratitude to my major Professor, Dr. Olaf
Mickelsen. His enthusiasm and love of learning have set a
constant goal for me.

My sincere thanks go to Dr. Dorothy Arata, an in-
spiring professor in my course work and Dr. Modesto Yang for
his willingness and help in instructing me in the methods
and practice of research.

To Michigan State University for my Graduate As-
sistantship, the National Cancer Institute for funds to carry
out this research (Public Health Service Research Grant
#CA-O7052—Ol), to Dr. Gert L. Laqueur, Chief, Laboratory
Experimental Pathology, NIAMD, NIH, Dr. C. K. Whitehair and
Dr. Gerald Mugera of the Veterinary Pathology Department,
M.S.U. for their cooperation in the pathological work in-
volved in these studies, and to Dr. J. C. Keresztesy, NIAMD,
NIH for preparation of the cycad materials, I express my

gratitude.

ii

To My Parents

TABLE OF CONTENTS

 

Page
I. REVIEW OF LITERATURE . . . . . . . . . . . . . 1
Literature Cited 9
II. STUDIES OF THE EFFECTS OF INGESTION OF CYCAS
CIRCINALIS BY RATS . . . . a . . . . . . . . 12
Introduction 13
Experimental 17
Results 18
Discussion 46
Literature Cited 51
III. ADDITIONAL RESEARCH PROJECTS . . . . . . . . . 54
Washed Cycad Flour 54
Introduction 54
General Procedure 54
Volitional Activity 55
Respiratory Infection 55
Reproductive Response 56
Tissue Preparation 60
Unwashed Cycad Flour 61
Introduction 61
Stability of Toxic Factor and Vari-
ations in Biological Response 61
Comparison of Toxic Response of
Cycasin and Unwashed Cycad Flour 62
Feeding Pregnant Rats 68
Feeding Weanling Rats 7O
Cycad Husks 71
Introduction 71
Effect of Environmental Temperature 72
Blood Changes 72

iv

Page

Cooperative Studies with the Veterinary

Pathology Department 73
Swine 73
Cow and Calf 77
Horses 8O

Table

LIST OF TABLES

Page

Composition of cycad flour and white wheat
flour . . . . . . . . . . . . . . . . . . . 15

Average weight changes of rats of different
strains fed unwashed cycad flour #623-6.
Five animals per group, average for 5
days . . . . . . . . . . . . . . . . . . . . 35

Weight changes (g) of Osborne-Mendel rats
from 2 sources fed unwashed cycad flour . . 36

Effect of age on weight changes (g) of
young Osborne-Mendel rats fed unwashed

cycad flour #623-6. Ten rats per group,
average for 5 days . . . . . . . . . . . . . 37

Summarizing table of short term bioassays
using unwashed cycad flour . . . . . . . . . 63

vi

Figure

LIST OF FIGURES

Growth depression of rats fed either of two
lots of unwashed cycad flour for 5 days

Calculated percentage cycasin in unwashed
cycad flour using weight depression curve
of rats fed unwashed cycad flOur for 5
days . . . . . . . . . . . . . . . .

Growth of rats fed cycad flour #628—1 incu-
bated for varying intervals at 37°C (5-day
bioassay) . . . . . . . . . . . . . . . .

Growth of rats fed heat treated, unwashed
cycad flour for 5 days . . . . . . . . . .

Growth of rats fed unwashed cycad flour
#623-6 stored at 5°C for varying inter—
vals (5—day bioassay) . . . . . . . . .

5-day body weight changes of rats fed un—
washed cycad flour in diets varying in
casein content . . . . . . . . . . . . .

Mortality of rats fed 2% unwashed cycad
flour for 18 or 25 days and/or 1%
thereafter . . . . . . . . .

Growth of animals fed washed cycad flour

% change in hemoglobin and hematocrit from

0 time 0 time animals fed 24% cycad husk
diet or control diet (pair-fed) . . . .

vii

Page

20

23

26

29

31

39

43

57

74

REVIEW OF LITERATURE

Poisonous plants are found in all parts of the world.
The tropical regions are virtual greenhouses for species
with toxic properties. Keegan and MacFarlane (1) list 8
families of plants, indigenous to Australia which are known
to be toxic when ingested and 181 species found on Guam
which are poisonous. These include plants which cause toxic
reactions after ingestion and/or on contact. Families of
such plants are characterized by distinctive poisonous
properties which may sometimes be affected by season,
climate, soil conditions and cultivation. Moreover, certain
parts of these plants may be highly poisonous whereas others
may be strictly harmless.

Mushrooms, cottonseed, soybean, the castor oil bean,
several species of the common sweet pea and certain plants
of the parsley family are some of the commonly known toxic
plants (2). A host of plants contain cyanogenetic glucosides
which may under certain circumstances cause hydrocyanic acid
poisoning. These glucosides have been found in corn,
sorghum, rough gram, field beans, black gram, kidney beans
sweet potatoes, lettuce, the seeds of some fruits, lima
beans and manioc. Only in these latter 2 is the content

of glucoside great enough to cause poisoning. Manioc is a

staple food in the diet of many Africans. In America manioc
is more commonly known as cassava or tapioca. Preparation
of the root starch of manioc involves soaking, fermenting or
boiling. Each treatment is considered sufficient to cause
complete loss of the toxicity responsible for liver, kidney,
skin and neural changes which result when unwashed manioc is
eaten (3). Growth inhibition in rats has recently been re—
ported by Kadake and Evans (4) when the raw navy bean was
fed as the source of dietary protein.

The genus Senecio of the aster family, contains 1250
species, widely distributed throughout the world and known
by a variety of local names most commonly "ragwort." Senecio
plants are found in parts of America, Europe, Africa, India,
Australia and Japan and have probably been the cause of more
livestock losses than all other poisonous plants combined
(5). The toxic prOperties of the pyrrolizidine alkaloids
produced in these plants have been the topic of extensive re—
search, particularly in Britain and Jamaica (6, 7, 8, 9).
Pathological changes seen in animals that have ingested
these compounds resemble closely those seen in animals fed
flour prepared from the nut of Cycas circinalis (10).

The first record of the toxicity of gygas is a note
in the diary of Cook dated 1770 in which he reports illness
in his men when they consumed C. media at Endeavour River,
Australia (l)° Since that time numerous reports relate the
ingestion of various parts of the cycad plants to illness in

both men and animals (11).

Cycads resemble the tree ferns in appearance and
represent an evolutionary transition between tree ferns and
Gymnosperms (12). They are of medicinal, economic and
nutritional importance in the tropics and subtropics where
the plant is indigenous. The nut, produced at the top of
the female plant is of most value (13). In Guam, juice
grated from fresh cycad nuts is a recommended treatment for
tropical leg ulcers, snake bites, open cuts and wounds.
Medicinal teas are also used (11). Limited use has been
made of the various parts of the plant in the manufacture of
alcohol, fertilizer, laundry starch and to a lesser extent
in construction (13).

A sturdy root structure enables the cycad plant to
withstand adverse climatic conditions. For this reason the
cycad is regarded as a valued emergency food in areas in
which it is indigenous. Flour milled from the starchy endo—
sperm is the form most commonly used. One of the values of
the nut is that when thoroughly dried it can be stored for
long periods. Hard dry pieces of sliced seeds are kept from
season to season. They are ground to a fine powder and used
in a variety of cooked dishes including tortillas, and
soups (11). Starches are also prepared from the roots and
stems of some species. "Arrowroot" has often been used to
refer to the starches made from the tuberous roots of certain
species. "Sago" appears to be restricted to starches ob-

tained from the stems (13). The proximate composition of the

flour prepared from the nut of Cycas circinalis is similar

 

to white wheat flour (Table l of Manuscript). In addition
to the flour and starches prepared from the plant, the husks
covering the nut are chewed fresh as the nuts are harvested
and later in dried form as a confectionary.

The cycad has long been recognized as toxic. It was
not until 1941 that Cooper (14) isolated from the seeds of

Macrozamia spirilis, a species of cycad native to Australia,

 

a compound that exhibited toxic properties.. Later this com-
pound was identified as a glycoside of primerverose and
methylazoxymethanol (MAM). A glucoside similar to that iso-
lated by Cooper was obtained from Cycas revoluta Thunbgby
Nishida in 1955 (15) and from_§ycas circinalis by Riggs in
1956 (16). The aglycone, MAM, CH34§=N-CH20H is common to all
toxic cycad glycosides identified and considered the toxic
factor of the plant (17). Methylazoxymethanol contains
nitrogen in linkages not previously found in a natural product
(11). On acid or enzymatic hydrolysis of the glycosidic bond,
the aglycone which is unstable decomposes spontaneously to
produce nitrogen, methanol and formaldehyde. Alkaline hydro-
lysis yields nitrogen, formic acid, cyanide and traces of
methylamine (18).

Through experience, the Guamanians have learned to
rid the nut of its toxicity by soaking it cut in eights, in

large vats of water. The soaking procedure varies. The

water may be changed often or relatively infrequently.

Guamanians report (19) that animals have died after drinking
the wash water from soaking nuts. This suggestion that the
toxic compound is stable in the water has not been borne out
in our laboratory work (Page 22, accompanying manuscript).
The soaking time varies from about 10 days to 3 weeks. The
nut pieces are spread to dry in the sun before being stored.
The nutritional safety of nuts so prepared is presently being
investigated.

The "unwashed" cycad causes mild and severe gastro—
intestinal symptoms when consumed. Such cases most frequently
occur during periods of extreme food shortage and can often
be traced to inadequate preparation of the nut. Symptoms
include swelling of the stomach, abdominal cramps, vomiting
and diarrhea. Headache, dizziness, depression and stupor
are often concurrent with the gastrointestinal ailments (11).
In 1 case, ingestion of inadequately prepared cycad caused
the death of 5 members of 1 family (20).

Nuts and leaves of the plant are eaten by animals as
well. In Australia, it is reported that animals graze se-
lectively on cycad. Some even become addicted. The losses
of cattle and sheep due to cycad poisoning, especially in
Australia, is a serious economic problem (21). One effect
on cattle and sheep is severe gastrointestinal disturbances,
the other is a partial paralysis. This paralysis in animals
is first apparent as a slight staggering gait and crossing of

the hind limbs in walking (11). Henderson (22) reports a

similar disturbance in oxen pastured in areas where the genus
Zamia grows in the Dominican Republic. Slightly more than 1%
of the work oxen in these areas are lost each year because

of this paralytic disease.

Numerous feeding trials, though sometimes poorly
controlled confirmed the development of the acute gastro-
intestinal symptoms and the chronic locomotor difficulties
(11). The course of the locomotor paralysis in cattle fol-
lows a characteristic pattern. It is progressive, ir—
reversible and exaggerated by exercise. Death occurs when
the animal can no longer move about to get food and water
(11,22).

Nishida (18) reported that mice injected subcu-
taneously or intravenously with crystalline cycasin, the

toxic glycoside isolated from Cycas circinalis, exhibited no

 

toxic symptoms, whereas 5 of 6 mice administered similar
amounts by stomach tube were dead in 36 hours. Similar
amounts given orally were lethal for the guinea pig.

Nishida (18) attributed the toxic manifestations appearing
after oral administration of cycasin to the absorption of
products released by the action of thejR-glucosidases present
in the gastrointestinal tract. Laqueur (24) has shown that
the acute effects of cycasin intoxication are completely
absent in germfree rats indicating that cycasin per se is

not injurious and that it is not cleaved to glucose and the

aglycone in the absence of bacterial enzymes.

Current research into the toxicity of the cycad de-
velopment from its implication as the neurotoxin responsible
for the high incidence of amyotrophic lateral sclerosis,
(ALS) among Guamanians. In Guam, the incidence, prevalence
and death due to ALS are approximately 100 times that in the
United States and Europe (25).

The disease affects the cells of the medulla and
cervical cord. It appears usually between the ages of
40 and 50. Weakness of the hands and arms, difficulty in
swallowing and talking, weakness and spasticity of the legs
are the first symptoms. In a fully developed case, the
muscles of the hands, arms, shoulders, pelvis, legs and
tongue become atrophied. The weakness of the legs appears
as a spastic gait. The course of the disease is rapidly
progressive and always fatal. In most cases death ensues in
2 to 5 years (26).

The increased incidence in ALS on Guam and the
Marina Islands prompted investigation into possible causa-
tive factors of the disease. The small number of families
and extensive intermarriage on Guam suggested that ALS may
be an expression of a recessive genetic trait. However no
genetic patterns became evident after investigation.
Environmental factors were also ruled out as being of pri—
mary importance (27).

In a broad nutritional survey (11) aimed at identi-

fying toxic foods which may be related to the development of

ALS, Cycas circinalis was suggested because of its associ-
ation with paralytic diseases in animals (19). However,
experiments with several species of animals failed to show
the neurotoxic effect of cycad ingestion (11). Even in
trials where cattle developed paralysis after being fed
cycad, microscopic examination of the brain and spinal cord
did not reveal any neurological basis for the disturbance
(28). Laqueur et a1 (10) report the absence of histological
lesions resembling ALS in the brain and spinal cord of rats
fed the flour of the Cycas circinalis nut. However, animals
fed small amounts of this flour over extended periods of
time developed benign and malignant tumors. These neoplasms
occurred mainly in the liver and kidneys and occasionally in
the lungs and intestine (10). These results leave little
doubt that the flour prepared from the fresh nuts of gygas
circinalis is carcinogenic and probably not directly associ-

ated with the development of ALS.

10.

ll.

12.

Literature Cited

 

Keegan, H. L., MacFarlane, W. V. 1963. Venomous and
Poisonous Animals and Noxious Plants of the Pacific
Region. The Pergamon Press, MacMillan Company, COpy—
right 1963.

Dubois, K. P. Geiling, E. M. K. 1959. Textbook of
Toxicology. Oxford University Press, Copyright 1959.

Jones, W. O. 1959. Manioc in Africa. Stanford
University Press, Copyright 1959.

Kadake, M. L., Evans, R. J. 1963. Growth Inhibition
in Rats Fed Raw Navy Beans. Fed. Proc. 22(2):551.

Campbell, J. G. 1956. An Investigation of the Hepa-
totoxic Effects in the Fowl of Ragwort with special
reference to the Induction of Liver Tumors with
Seneciphylline. Proc. Royal Soc. Edinburgh, LXVI:lll.

Hill, K. R. 1960. Discussion on Seneciosis in Man and
Animals. Proc. Royal Soc. Med., 53:281.

Campbell, J. G. 1960. Variations of Liver Function in
Immature Fowls and Susceptibility to Seneciphylline.
J. Path. Bact., 80:399.

Schoental, R., Magee, P. N. 1959. Further Observations
on the Sub—acute and Chronic Liver Changes in Rats
After a single does of Various Pyrrolizidine (Senecio)
Alkaloids. J. Path. Bact., 78:471.

Schoental, R. 1960. The Chemical Aspect of Seneciosis.
Proc. Royal Soc. Med., 53:284.

Laqueur, G. L., Mickelsen, O., Whiting, M. G., Kurland,
L. T. 1963. Carcinogenic Properties of Nuts from
Cycas Circinalis L. Indigenous to Guam. J. Natl. Cancer

 

Inst., 31:919.

Whiting, M. G. 1963. The Toxicity of Cycad. Economic
Botany, 17:271.

Missouri Botanical Garden Bulletin. 1955. 43:65.

13.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

24.

25.

10

Thieret, J. W. 1958. Economic Botany of the Cycads.
Economic Botany, 12:3.

Cooper, J. M. 1941. Isolation of a Toxic Principle
from the Seeds of Macrozamia Spirilis. J. Proc. Royal
Soc. New South Wales, 74:450.

 

Nishida K., Kobayashi A., Nagahama, T. 1955. Cycasin,
A New Toxic Glycoside of Cycas Revoluta Thunb. Bull.
Agr. Chem. Soc. Japan, 19:77.

Riggs, N. V. 1956. Glycosylazoxymethane, A Constituent
of the Seeds of Cycas Circinalis L. Chem. and Industry,
35:926.

Matsumoto, H. 8., Strong, F. M. 1963. The Occurrance
of Methylazoxymethanol in Cycas Circinalis L. Arch.
Biochem., 101:299.

Nishida, K., Kobayashi, A., Nagahama, T., Kojima, K.,
Yamane, M. 1956. Cycasin, A New Toxic Glycoside of
Cycas Revoluta Thunb: IV Pharmacological Study of

 

Cycasin (In Japanese) Seikagahu, 28:218.

Whiting, M. G. (personal communication) NIH, Bethesda,
Md.

Hiroyshi, T. 1956. Cycad Poisoning on Island of
Miyako. Okinawa Times, Dec. 13.

Seddon, H. R., Belschner, H. G., King, R. O. C. 1931.
Poisoning of Sheep by the Seeds of Burrawang (Macro-
zomia Spirilis) Vet. Res. Report #6. June. Dept.
Agr., New South Wales, Sydney, Australia.

Henderson, J. 1964. (personal communication) Confer—
ence--Current Progress in Studies of Cycad Toxicity
with particular reference to Neurotoxic Properties.
NIH, Oct. 26, 1964.

Hall, W. T. K. 1956. A Note on Macrozamia and
Xanthorrhoea Poisoning of Cattle, Australia Vet. J.,
July: 173.

Laqueur, G. L. 1964. Carcinogenic Effects of Cycads
and Cycasin in Rats. Fed. Proc. in the press.

Lessell, S. 1962. (personal communication) NIH,
Bethesda, Md.

26.

27.

28.

11

Cecil, R. L., Loeb, R. F. 1955. A Textbook of Medicine.
W. B. Saunders Co. Philadelphia. p. 1459.

Kurland, L. T. 1962. (personal communication) NIH,
Bethesda, Md.

Hall, W. T. K. 1957. Toxicity of the Leaves of Macro—

azamia Spirilis for Cattle. Queensland J. Agr. Sci.
14:41.

STUDIES OF THE EFFECTS OF INGESTION OF

CYCAS CIRCINALIS BY RATS

M. E. Campbell, 0. Mickelsen, M. G. Yang,
G. L. Laqueurl and J. C. Keresztesy2

Department of Foods and Nutrition
College of Home Economics
Michigan State University

East Lansing, Michigan

This investigation was supported by Public
Health Service Research Grant No. CA—07052-01 from
the National Cancer Institute

lChief, Laboratory of Experimental Pathology

2Chief, Section of Fractionation and Isolation
National Institute of Arthritis and Metabolic Diseases

National Institute of Health

submitted to Journal of Nutrition for publication

Introduction

 

Cycas circinalis L. is one of 9 species of cycads
growing in tropical or subtropical areas. Cycads resemble
tree ferns and represent an evolutionary transition between
ferns and Gymnosperms or evergreens (1). They are of medi—
cinal, economic and nutritional importance in the areas
where they are indigenous (2). Although some species of
cycad are widely distributed, Cycas circinalis L. is indi-
genous to Guam and Oceania.

As early as 1770, field observations suggested that
cycads contained toxic substances. Reports of these obser-
vations indicated gastrointestinal and paralytic symptoms
developing in animals and man after ingesting parts of the
plant (3, 4). The first sign of paralysis in animals is a
staggering gait with a crossing of the hind limbs when rest—
ing. In this position, the animal sways from side to side,
hence the term "wobbles" used by the Australians to describe
this condition (3). Great losses of cattle and sheep have
been reported from regions where cycad grows on grazing
lands (5).

Some of the first animal studies, although often
poorly controlled, confirmed the development of gastroin-

testinal symptoms and locomotor disturbances (3). Severe

13

14

liver damage was a common pathological finding. In some
animals, chronic feeding of cycad resulted in a locomotor
disturbance that was progressive, irreversible and was ex-
aggerated by exercise. However, no neurological basis for
this paralysis was found (6).

Present interest in Cycas circinalis stemmed from an
apparent correlation between its use as a human food and a
high incidence on Guam of amyotrophic lateral sclerosis.
This disease is 100 times more prevalent on Guam than in the
United States or Europe (3). The disease is characterized
by neural and muscular degeneration, appears usually between
the ages of 40 and 50, progresses rapidly and terminates
fatally within 2 to 5 years (7).

The use of cycad by the natives on Guam and other
Southwestern Pacific Islands stems from the fact that this
plant survives adverse climatic conditions. This makes it
one of the few foods available during droughts and following
devastating typhoons. The nut meat is the part of the plant
that is most frequently used by man. Since its toxicity is
well recognized, it is routinely soaked in water prior to
use. The nut (approximately 4 cm in diameter) is quartered
and the center, or nut meat, soaked in vats of water. The
water may be changed frequently during soaking which lasts
from a few days to 3 weeks. The nut slices are dried in the
sun and then ground into flour. The latter is used for

thickening soups, puddings, porridge and in tortillas (3).

15

The proximate composition of cycad flour is similar
to wheat flour (table 1). The 34% not identified is thought
to be carbohydrate. However, with the exception of glucose
and fructose, the other carbohydrates are present in trace

amounts only.

Table 1. Composition of cycad flour and white wheat flour.

 

 

 

Cycad Flour White Wheat Flourb
(%)
Nitrogen 2.0 1.8
Water 6.5 12.0
Fat 0.8 1.0
Ash 2.6 0.4
Carbohydrate (known) 45.0a 76.1

Unknown 34.0

 

aThe known carbohydrates as a percentage of cycad
flour are: free glucose 1.2 - 2.1, glucose (freed by acid
hydrolysis) 39.0, free fructose 0.7 - 0.9, fructose (freed by
acid hydrolysis) 1.4, cycasin 2.0 and trace quantities of
xylose, primeverose (?), myo-inositol, sequoyitol and pini-
tol (personal communication from Drs. N. K. Richtmyer and
S. S. Chernick, National Institute of Arthritis and Meta-
bolic Diseases, National Institutes of Health). Determin—
ation of total carbohydrate by the anthrone method (8)
showed cycad flour was 52% carbohydrate.

bsee reference (9).

In 1941, Cooper isolated from Macrozamia spirilis, a

species of cycad indigenous to Australia, a glycoside which

16

was lethal to animals (10). Cycasin, a glycoside similar to
the one isolated by Cooper except for the sugar moiety, was
isolated from Cycas revolunta Thunb. by Nishida in 1955 (11)
and from Cycas circinalis L. by Riggs in 1956 (12). The

aglycone common to these glycosides is methylazoxymethanol
o

CH3—NsN-CH20H. It is considered to be responsible for the
toxicity of the glycosides (13).

Evidence suggests that the aglycone is toxic only
after hydrolysis of the glycoside by bacterial Af—glucosidases
in the intestinal tract. Nishida (14) noted that toxic
symptoms appeared only after oral ingestion of the glycoside
but not following intravenous or subcutaneous injection.

The absence of acute toxicity in germfree rats fed cycasin
confirms this hypothesis (15).

To determine whether there is any experimental basis
for implicating cycad in the etiology of amyotrophic lateral
sclerosis, unwashed cycad was fed to various species of ex—
perimental animals. The present paper reports the stability
of the toxic factor(s) of cycad and variations in the re-
sponse of rats of different sexes, ages, strains and colonies.
Reported, as well, are the effects of dietary changes and
discontinuation of cycad feeding on the toxic response.

A previous report of this work (16) indicated that
the chronic ingestion of cycad produced no central nervous
system lesions in rats. Instead, neoplastic lesions de—

veloped in the liver and kidneys.

17
Experimental

Cycad Materials

Cycad nuts were shipped to our laboratory immediately
after collection from various parts of Guam. Each lot of
nuts was coded with the year, month and day of delivery (e.g.
623—6). These materials had not been soaked as described
earlier, and will be referred to as unwashed cycad. The
latter material was chosen because it was thought to contain
a higher concentration of the toxic factor and thus results
obtained would be more definitive. The nuts were husked,
cut open, thinly sliced by machine and dried under vacuum at
400C. The dried slices were ground into flour in a Wiley

Mill.

Bioassay

Sprague—Dawley and Osborne—Mendel male weanling rats
were used except where indicated. Previous to the feeding
of experimental rations, all animals were fed a basal grain
ration until a weight increase of 5 to 6 grams per day was
achieved. The average body weight of the animals at the be-
ginning of the experimental feeding trials was approximately
80 grams. In most studies, 10 animals comprised each experi—
mental group. In some of the preliminary work, 3 or 5
animals were used. All were housed individually in suspended

wire cages. Feed and water were given ad libitum.

18

Natural grain rations were fed as the basal diet.1

Cycad materials were thoroughly mixed with appropriate pro-
portions of the basal diet and refrigerated. Prior to feed—
ing, all cycad materials were stored at SOC.

Growth rates and liver lesions were used to indicate
the relative toxicity of each new lot of cycad flour.
Weights were recorded on the first and fifth day of the
assay period. On the fifth day, the animals were etherized
and sacrificed. The livers were removed, fixed in 4%
acetate-buffered—formalin and later examined for pathological

lesions. Some animals were maintained for longer periods.
Results

General

Animals fed the unwashed flour as a percentage of
their basal ration gained less than controls fed basal ration

only. Growth depression of animals varied depending upon the

 

1A modification of the Animal Nutrition Research
Council Reference Chick Diet (17), the percent composition of
this grain ration was: ground corn 60.7, soybean meal (50%
protein) 28.0, alfalfa meal (17% protein) 2.0, fish meal
(12.5% protein) 2.5, dried whey (67% lactose) 2.5, limestone
(38% Ca) 1.6, dicalcium phosphate (18.5% P, 22-25% Ca) 1.75,
iodized salt 0.5. Supplementary minerals, vitamins and anti-
biotics were added to provide per kilogram of feed: (in mg)
Mn 121, Fe 95, Cu 7, Zn 4, I2 4, Co 2, choline chloride 400,
calcium pantothenate 6, riboflavin 3, niacin 33, menadione 2,
DL Methionine 500, penicillin 2, streptomycin 8, arsenilic
acid 968, (in/ag) vitamin B12 7, (in International units)
Vitamin A 8010, Vitamin D2 750, Vitamin E 5 (Alpha Tocopherol
Powder, Nutritional Biochemical Corporation, Cleveland, Ohio).

19

lot of flour used, but, all flours caused a linear response
when weight changes were plotted logarithmically against the
percentage of unwashed cycad flour included in the ration
(figure 1). We have plotted growth depression for all bio-
assays as the logarithm of the change in weight + 40 (to
avoid negative numbers) and the corresponding actual body
weight change (in g) on the ordinate. Zero percent cycad
indicates the growth response of control rats. Vertical
lines are Mantel's standard errors (18). Data were analyzed
by analysis of variance (l9) and mean differences by Duncan's
Multiple Range Test (20).

Decrease and loss of cytoplasmic basophilia from the
centrolobular zones and necrosis of individual liver cells
in these zones are characteristic of the liver lesions seen
in the animals fed experimental rations. With increasing
concentrations of cycad flour, there was a corresponding in-
crease in the severity of the liver damage. These lesions
are identical with those reported by Laqueur et a1 (16) as
being the early changes characteristic of cycad intoxication.

A considerable amount of the present work has in—
volved the use of cycasin, the major glycoside in the cycad
flour. Preliminary studies suggest that most of the toxic
symptoms associated with the feeding of unwashed cycad can
be attributed to cycasin. Body weight changes for 90 male
Sprague-Dawley rats, fed various levels of unwashed cycad

flour or cycasin in a 5—day bioassay, suggest that the flour

20

FIGURE 1

GROWTH DEPRESSION OF RATS FED EITHER OF

TWO LOTS OF UNWASHED CYCAD FLOUR

FOR 5 DAYS

21

AmEmuw CHV wmcmnu ucmflwz hpom Hmsuofl

 

 

 

 

 

8 9
8 l 2 l 2 5 4 o o
o o o o 5 o o u l 4
O 3 6 O . O 4 8 l l
3 2 l l 4 _ _ _ _ _
. T h p p . . . . p
O
61
._
38
22
66
##
._. -4
_
.
_
O
r.3
T2
11
n .8 % 6 5 4
2 1 1 11 .1 11 1

on + mEmuw CH mmcmno unmflwzv moq

% Cycad Flour in Basal Ration

22

contains approximately 2.3% cycasin (dry weight basis)
(figure 2). The break in the curve at the 1% cycad level is
unexplainable. This was the first assay (in approximately
25) where the curve showed this break. Apart from that, the
slope of the main part of this curve parallels that of the

previous assays run under comparable conditions.
Stability of Toxic Factor

Water Treatment

Reports from Guam suggest the "wash water" from soak-
ing nuts is lethal to animals (21). This would indicate the
toxic factor is water soluble, can be leached out of the nut
pieces and is stable in solution. To test this hypothesis,
624 g of unwashed cycad nut slices, of known toxicity (see
#623-6, figure 1), were soaked in 1.5 liters of tap water at
37°C for 5 days. The solids were removed by filtration,
dried at room temperature and ground. The filtrate was
concentrated under vacuum to 66 m1. Diets containing either
5% ground solids, an aliquot amount of the concentrated fil-
trate, or both, were fed to groups of 10 weanling rats.
Growth responses for the 5-day assay averaged 31, 32, and
33 g respectively. These increases were similar to gains of
control animals, indicating the absence of the toxic factor
from both the residue and the filtrate prepared in our

laboratories. These data, contrary to reports from Guam,

23

FIGURE 2

CALCULATED PERCENTAGE CYCASIN IN UNWASHED CYCAD

FLOUR USING WEIGHT DEPRESSION CURVE OF RATS

FED UNWASHED CYCAD FLOUR FOR 5 DAYS

24

 

 

 

 

 

 

 

2.
o O cycad
+ +' cycasin
l.% ~39.4
P
t30.8
81°8 0 b23ol
d‘ .
+
m —16.2
E
S
(91.1 F10.l
c
~H
g L4.5
c
2
01.6 .5 ,—o.2
4.) U)
g, 8 s
-:--l >1 'P‘l
(D o g '-4.5
E F. 0
Q4 >1
m1,5 9 U --8.4
S o s
o 91
E W
o
o
m
1.4
1.3 T . IAML I :y r 1
l 1.5 2 3 4

% Cycad Flour 628-10 in Basal Ration
Calculated % Cycasin in 628-10 Cycad Flour

2.28 for animals fed 400 ppm cycasin

2.35 for animals fed 600 ppm cycasin

(in Grams)

Actual Body Weight Change

25

suggest a degradation of the toxic compound has occurred
during the soaking process.

To study the rate of loss of toxicity, pastes made
from unwashed cycad flour and water were incubated at 370C
for varying lengths of time, dried under vacuum at 400C, and
ground into flour. The growth of animals fed a diet contain-
ing 15% non—incubated flour was less than that of animals
fed diets 15% of which was the incubated flour. The treat-
ment caused a progressive decrease in the toxicity of the
pastes with time. Large volumes of gas were generated over-
night and all toxic activity was lost some time prior to 90
hours of incubation (figure 3). The gases evolved are be-
lieved to be breakdown products (probably nitrogen) associ-
ated with the destruction of toxicity. However, not all
pastes, made from different lots of unwashed flour, similarly
incubated, produced gas. Pastes made from washed (i.e.
soaked) cycad flour did not evolve gas with or without the
addition of the crystalline glycoside, cycasin.

That the toxic component of cycad nuts is destroyed
during incubation in the moist state is evident. Experiments
are presently underway to test the nutritional safety of the

cycad prepared for food by the natives on Guam.

Heat Treatment

Cycad flour is used chiefly as an ingredient in the

cooked dishes of the Guamanian people. The effect of heat

on the stability of the toxic factor was next investigated.

26

FIGURE 3

GROWTH OF RATS FED CYCAD FLOUR #628—1
INCUBATED FOR VARYING INTERVALS AT

37°C (5-DAY BIOASSAY)

Log (Weight Change in Grams + 40)

1.6

27

 

 

P30.8

t23.1

’16.2

(~10.1

” -11.8

.-l4.9

 

 

Hours of Incubation at 37°C

/
/
/
/
/
/
/
/
/
/.
l .
/
/
/
/
/
/
/
/
/
I/
/
/l
/
/
/
/
/
1'0 2'0 3'0 40 5'0 6'0 7'0 8'0 9'0

28

Toxic unwashed flour (lot #637-3) was cooked at
900C for 1 hour with 9 volumes of tap water, dried in thin
layers at 370C for 30 hours and ground in a Wiley Mill. An
amount of the same flour was heated at 900C for 1 hour with-
out the addition of water. The growth response of animals
fed either of these heat treated flours at levels of 2, 3, or
4% of the ration was in most cases, similar to control
animals fed only the basal ration (figure 4). With the ex-
ception of the group fed 4% cooked flour (moist heat), the
livers from these animals were microscopically normal. This
latter group showed loss of some basophilia from the centro-
lobular areas. These data would indicate the toxic factor
in unwashed cycad flour is almost completely destroyed by
moist or dry heat at 90°C. This instability of the toxic
factor to heat adds a probable margin of safety to the con—
sumption of washed cycad flour when it is used in cooked

foods.

Storage

Growth curves from successive animal feeding trials
indicated a loss in the toxicity of the cycad flour during
storage. Flours stored at -200C retained more toxicity than
flours stored at 5°C. One lot of unwashed flour (#623—6),
stored at 50C, was assayed 4 times over a period of 5 months.
Figure 5 represents the data obtained from the first and

fourth assays. The curve from the second and third assays

29

FIGURE 4

GROWTH OF RATS FED HEAT TREATED, UNWASHED

CYCAD FLOUR FOR 5 DAYS

31

FIGURE 5

GROWTH OF RATS FED UNWASHED CYCAD FLOUR #623-6
STORED AT 5°C FOR VARYING INTERVALS

(S-DAY BIOASSAY)

32

 

2.0
X'-———X 5 Months
F—-———o 1 Day
1.9d -———————- 10 Months
~30.8
8 1. r23.1
<r
+
(D '16.2
E
S
0 1. _10.l
C.‘
H
g *4.5
C1
1:"
Q 1. P-O.2
4.:
8
-$ )—-4.5
E
01 1. s-8.9
O .
r—l
\
\ lFll 3
\
\
l \ '-14.9

 

 

 

 

1.3 I I I T

1 2 3 4 5

% Cycad Flour 623-6 Added to Basal Ration

33

lies between those shown. A fifth assay at Michigan State
University laboratories, after 10 months storage (figure 5),
indicated the toxicity of the flour was greater than in the
initial assays at NIH. To study this apparent increase in
toxicity of 1 lot Of flour, the effect of different rations
and different strains and age of rats on the bioassay was

investigated.

Rations

Various commercial rations were fed as the basal
diet. The very poor growth of control rats receiving 1
commercial ration suggested some dietary abnormality. In-
quiry revealed that this ration2 contained a coccidiostat3
which apparently reduced the food intake and thereby the
growth of the rats. Apart from this complication, 3 other

grain rations used4 had no effect on the bioassay.

Strain, Sex and Colony Variations

Body weight losses of Osborne—Mendel rats fed
various levels of unwashed cycad flour indicated that this

strain was slightly more susceptable to the toxicity than

 

2Chick Startena — Ralston Purina Co., St. Louis,
Missouri.

33,5-dinitro-o-toluamide, Zoalene - The Dow Chemical
Company, Midland, Michigan.

4

M—1 (see footnote 1), Rockland Mouse/Rat Diet,
Tehlad, Inc., Monmouth, 111., Nutritionally Adequate Natural
Grain Pig Ration prepared locally, Michigan State University
Pig Grower Ration #1.

34

were rats of the Sprague—Dawley strain. This was more
noticeable in the female than in the male animals (table 2).
In addition, male Osborne-Mendel rats obtained from the
colony at NIH lost more body weight than did rats of the

same strain purchased from a commercial outlet5 (table 3).
These differences however, were not statistically significant

(P 7 0.05) .

yAge of Animals

Newborn rats appeared to be much more susceptable
to cycasin toxicity than weanling animals. In 1 assay,
when neWborn pups were permitted to suckle a dam fed a
natural grain ration containing 400 ppm cycasin, all pups
were dead by the fifth day. However, when the same ration
waS‘fed to weanling rats, 50% of them survived for more than
7 months. In a third trial weanling animals 25 days Old
(759) lost a greater percentage of their body weight than
animals 37 days old (130g) when both groups were fed diets
containing 2, 3, or 4% unwashed cycad flour (table 4). Al-
though these differences were not statistically important
(P;> 0.05), the 3 trials showed a similar trend, indicating
that younger animals are more susceptible to cycad toxicity

than are older ones.

 

5Osborne-Mendel rats purchased from Camm Research
Institute, Wayne, New Jersey. Sprague-Dawley rats purchased
from Spartan Research Animals, Inc., Haslett, Michigan.

35

 

 

 

Table 2. Average weight changes of rats of different
strains fed unwashed cycad flour #623-6. Five
animals per group, average for 5 days.a
Strain Diet Male Female

Osborne— Weight gain (g) Control 29.6 24.2

Mendel 2% cycad 11.2 6.4

Weight decrease
(% of Control) 62.2 73.6
Sprague- Weight gain (g) Control 34.6 26.4
Dawley 2% cycad 14.2 9.4
Weight decrease
(% of Control) 59.0 64.4

 

P<0.0l:

significant, P > 0. 05 .

aLevels of cycad fed were significantly different at
comparisons of sex and strain differences were not

36

Table 3. Weight changes (g) of Osborne-Mendel rats from 2
sources fed unwashed cycad flour.a

 

 

 

% Cycad NIH Camm Research
in Diet Animals Institute Animals
0 31.1 32.4
1 b 29.5
2 - 0.1 4.0
3 b — 6.7
4 -l6.4 —12.3

 

a2 and 4% cycad were significantly different from
each other and from control P<:0.01, differences in colonies
were not significant, P >0.05.

bThese levels were not used for NIH Animals.

37

Table 4. Effect of age on weight changes (g) of young
Osborne-Mendel rats fed unwashed cycad flour
#623-6. Ten rats per group, average for 5
days.a

 

 

 

Age and weight

       

 

at start of Expt. 25 Days 37 Days
% Cycad (75 g) (130 g)
in diet
0 28.2 32.4
2 2.0 4.0
3 - 6.2 - 6.7
4 -10.3 -12.3

 

aAll levels of cycad were significantly different
from controls P<f0.01, 3 and 4% cycad were different from
2% cycad P<:0.01, responses due to age were not signifi-
cantly different, P) 0.05.

38

Dietary Effects

 

Proteins provide the amino acids essential for cell
regeneration and they are generally regarded as being po-
tentially able to ameliorate the effects of toxic substances,
especially those effecting the liver (22). Since one Of the
primary effects of cycad ingestion is centrolobular liver
necrosis, rations containing different levels of casein were
studied for their effect on cycad toxicity. Zero, 1, 2 or
3% unwashed cycad was incorporated into 3 purified rations
containing either 12, 18 or 24% casein.6 These 12 diets
were fed to groups Of 5 weanling Sprague—Dawley rats for 5
days. All animals were fed the amount of feed eaten by the
rats receiving the ration containing 3% cycad flour and 12%
casein. Because the food intake was restricted, all animals
lost body weight. The degree Of weight depression, in the
animals fed the various diets, showed the 24% casein diet
reduced the toxic response in rats when this diet contained
up to 2% unwashed cycad flour (P<f0.01). The reverse was
true when the dietary levels exceeded 18% casein and 2%
cycad (P<f0.01). Diets containing 12% casein seemed to pre-
vent an increase in the toxic response when the intake of

cycad flour was increased (figure 6). A 2-way analysis of

 

6 . . . .
The source of casein was High Protein casein, General

Biochemicals, Chagrin Falls, Ohio. Each diet contained in
%: 4, Wesson Salt Mixture, a modification of Osborne-Mendel
Salt Mixture, Nutritional Biochemicals Corp., Cleveland,
Ohio; 2.2, vitamin supplement, Vitamin Diet Fortification
Mixture, Nutritional Biochemicals Corp., 5, corn oil and
sucrose to make 100.

39

FIGURE 6

5-DAY BODY WEIGHT CHANGES OF RATS FED

UNWASHED CYCAD FLOUR IN DIETS VARYING

IN CASEIN CONTENT

5-Day Assay

Weight Loss in Grams —-

_12 ..

 

40

o—————o

x-—-—-x

H-fi

24% Casein
18% Casein
12% Casein

 

 

I

1

T I

2 3

% Cycad Flour 632-4 in Purified Diets

Percent Cycad

LUMP-‘0

Percent Casein

 

12 18 24
1.6 1.4 1.4
0.8 2.0 1.0
1.2 1.2 1.0
0.8 1.0 0.6

41

variance indicated that regardless of the level of protein
(casein) in the diets, the weight depression caused by the
intake of 3% cycad was significantly different from that
caused by 0 or 1% cycad (P<:0.0b data broken down according
to levels of casein are not shown, but can be, calculated
from figure 6).

Methionine, because of its lipotropic activity and
sulphydryl group has been considered the effective principle
in the beneficial role of high protein diets in modifying
experimental hepatic toxicities (22). Preliminary investi-
gation with groups of 3 rats indicated that the addition of
1.0 or 0.5% DL methionine, to a ration containing 2% unwashed
cycad flour, exerted a beneficial effect on the growth Of the
animals but, a similar fortification of a 3% unwashed cycad
diet with methionine had a negligible effect. In a later
study, using groups of 10 weanling rats, fortification of a
2% unwashed cycad diet with 1% DL methionine7 exaggerated
the growth depression over the 3—week experimental period.
Weight gains for animals fed 2% cycad averaged 124 g, those
fed the 2% cycad fortified with methionine gained 110 g, and
control animals 139 g. The mean weight changes calculated
from these data are significantly different from each other
(P<:0.01). Thus, methionine, added to a ration containing

adequate protein, has no beneficial effect on the growth

 

7Nutritional Biochemicals Corp., Cleveland, Ohio.

42

response of rats receiving cycad and at a level of 1% may
actually accentuate the toxicity. No attempt was made to
establish if this level of methionine was toxic per se. Our
interest was in whether added methionine would reduce the

toxic response Of animals fed cycad.

Irreversibility of Toxic Response

 

Our next investigation was aimed at determining
whether the lesions induced by the intake of unwashed cycad
were permanent and progressive or reversible. For this
purpose 2 groups Of male, weanling, Osborne-Mendel rats were
fed diets containing 2% unwashed cycad flour. At the end of
18 and 25 days the cycad content Of the diet of each group
was reduced to 0 (19 animals) or 1% (26 animals) respectively.
Dietary changes were made at these times because the animals
were rapidly losing body weight. It was reasoned that if
this weight response was reversed when the diet was changed,
there would be no doubt as to the body's ability to overcome
the toxicity. The animals showed a slight weight gain after
the dietary change but the mortality of both groups indicated
the damage at this stage was permanent. The group fed the
1% cycad ration for the latter part of the experiment had
96% mortality while those fed the cycad-free ration for this
period had 79. A third group fed the control ration through—
out the study had only 13% mortality (figure 7). These re-

sults indicate the irreversibility of the damage initiated

43

FIGURE 7

MORTALITY OF RATS FED 2% UNWASHED CYCAD FLOUR

FOR 18 OR 25 DAYS AND OR 1% THEREAFTER

44

ucmEflummxm co mxmn

 

 

om pm 90 mm as mm mm ca
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haco Houucou 4|" Ila
Owning Km 0 . \\\\ 0
¥\ T...
emoso so 68% RN xiii}... \\ 1 w
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633 RA 685 8.1-1-1 \\\\ \ O
..\- . \. a m
\ \ \\\ . \\ m... w m.
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Em

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45

in rats fed unwashed cycad flour for as short a time as 18

days.

Absence of Cycad Toxic Factor in Liver

 

Since the liver plays an important role in the de-
toxification of harmful substances and is one of the primary
targets of cycad toxicity, the possibility that this organ
accumulates the toxic substance was investigated. The liver
was removed from a heifer that had been fed a calf starter
ration containing 2% unwashed cycad flour. After 43 days Of
feeding, the heifer had gained very little weight and showed
symptoms suggestive of neurological disturbances (occasional
difficulty in walking and standing). The heifer was sacri—
ficed at this time because it appeared close to death. The
liver was freeze-dried and ground to a fine powder. Groups
Of 10 weanling rats were fed this dried liver or liver from
a normal heifer at levels of 2, 5, or 10% Of their basalgrain
ration. Weight gains over a 5-day assay period were similar
for all groups. Average gains (in g) for animals fed the
liver from the cycad-fed heifer vs. those for the animals
fed the liver from a control heifer were respectively 31.8
vs. 32.3 (2%), 32.9 vs. 24.9 (5%), and 28.4 vs. 30.2 (10%).
These data suggest that the compound responsible for the
toxicity of cycad was not stored in the livers of the

animals fed the unwashed cycad.

46
Discussion

The reliance on the growth bioassay in estimating
the toxicity of cycads under various conditions stems from a
fairly good correlation between growth rate and the degree
of liver lesions (15). Although animal growth followed the
food consumption pattern, records indicated the experimental
animals consumed near normal amounts of food during the
first day but markedly less from the second day onward (un-
published data). Liver changes were recognizable as early
as 24 hours after cycad feeding was initiated (15). Food
consumption merely reflected this rapidly developing meta-
bolic disturbance.

Bioassays have shown that the toxicity Of the cycad
nuts is related to their stage Of maturity. The green nuts
are more toxic than the riper or brown-colored ones. There
is evidence that this toxicity is attributable to the glyco-
side, i.e., cycasin content of the nut (unpublished data).
Comparative assays suggest that the flour prepared from the
green cycad nuts contains as much as 2 to 4% cycasin.
Whether all the toxicity is traceable to the glycoside has
been questioned by Matsumoto and Strong (12) who secured evi—
dence for the presence of the free aglycone in Cycas circin-
alis. The aglycone, methylazoxymethanol, is a water-soluble
compound released from cycasin by the action Of a xg-glucosi-

dase. According to Nishida et a1 (7), it is the aglycone

47

which is responsible for the toxicity of the cycad plants.
The presence of.4?-glucosidases in the intestine of the rat
and their absence in liver and other tissues has been pro-
posed by Nishida et al (7) as the explanation for the Obser-
vation that cycasin is toxic only when given orally. In-
jection of this compound over short periods of time has been
reported to produce no symptoms of poisoning (7). The ab-
sence of a toxic response in germfree animals fed cycasin and
the near total recovery of the ingested compound in the
urine and feces of these animals confirm the suggestion that
bacteria bring about the cleavage Of cycasin (15).

The toxic material in the dry unwashed cycad flour
loses approximately half Of its toxicity when stored for 6
months at temperatures near -20°C. The cycad flour appears
to contain enzymes able to destroy the toxic compound (figure
3). The stability of the cycad material during storage
probably depends upon the activity of these enzymes.

The fact that the rate of destruction of the toxic
factor, as measured by differences in weight gain shows a
logarithemically linear progression with time suggests a
first—order reaction (figure 3). Despite the implication
that the washing process carried out on Guam involves simply
a leaching of the toxic factor, it is likely that an enzy-
matic or chemical degradation of the toxic factor occurs.
Temperatures on the island, ranging from 20 to 320C (23),

would favor enzymatic reactions. In addition, the physical

48

barrier to water penetration, (these slices are approximately
1 cm thick and show little sign of water uptake) argues
against the leaching process as being effective in the re-
moval of the toxicity. The foam reported on the surface Of
the "washing" vat suggests the compound undergoes some degra-
dation involving the loss of a gas, possibly nitrogen, when
the nuts are kept in water. The toxic factor is labile to
both moist and dry heat. Our Observations suggest that dry
heat is slightly more effective in destroying the toxicity
than is moist heat.

Protein is generally regarded as being protective
against liver injury (22) and high dietary intakes Of pro-
tein are known to modify the effects of some toxic chemicals
(24, 25). Such was the case in our experience when the diets
contained less than 2% unwashed cycad flour but at levels
above 2% this beneficial effect was not evident. In this
case, a 12% casein diet, although well below the range recom—
mended for growing rats (26), exerted a slight beneficial ef-
fect on the growth depression resulting from cycad ingestion.

Methionine is beneficial in the treatment of some
experimental hepatic injuries especially when there has been
protein depletion (22). However, the growth depression of
rats increased when 1% DL methionine was added to experi—
mental diets containing cycad. Harper (27) reports a methio-
nine toxicity in rats fed 2.5% DL methionine in both adequate

and inadequate protein diets. Our data would indicate a

49

synergistic action Of the cycad toxic factor and added
methionine.

Variations in the response Of animals of different
strains and colonies are not unexpected. Elevenfold differ-
ences in resistance to thiourea were reported (28) when rats
of the same strain but from different colOnies were ad-
ministered this chemical. Greater sensitivitycnr resistance
in animals Of 1 sex for some drugs and toxins undoubtedly re-
sults from metabolic and hormonal differences (25). However,
in the present study, the toxicity of cycad did not vary sig-
nificantly in rats of different sexes, strains or colonies.

The rapid turnover Of body tissue and cellular com-
ponents may account for the increased sensitivity Of younger
animals to the toxicity Of cycad. In some drug and chemical
intoxications resistance is better in younger animals (28)
but these observations are the exception rather than the
rule.

The liver cells reacted almost immediately to un-
washed cycad flour--changes appear as early as 24 hours
after ingestion (15). The cumulative response caused by the
ingestion of the flour for 18 days resulted in damage that
was irreversible. Feeding trials indicated however, that
the toxic compound is not stored in the liver of animals fed
cycad. Thus, the question arises whether after the initial
liver damage takes place, detoxification of the toxic com—

pound occurs in the liver or at a different site.

50

All of these observations can be of practical value
when considered in terms of the consumption of cycad foods
by people. Experiments are underway to study the nutri—
tional safety of these foods as they are eaten by Guamanians.
Results Of these trials should be considered in relation to
the response and stability reported in this paper. Our
observations raise a number of questions as to the impli-
cations Of cycad consumption by humans. A considerable
amount of additional information is required before defini-
tive answers can be given as to whether cycad consumption by
humans should be discouraged. There is an urgent need to de-
.termine how much cycad is consumed by humans in those parts
of the world where the plant is indigenous and to Obtain
some indication as to the age groups who use it. It would
also be desirable to carry out an epidemiological study of
the incidence Of various neoplastic diseases among these

same people.

10.

ll.

12.

13.

51

Literature Cited

 

Missouri Botanical Garden Bull. 1955. 43:65.

Thieret, J. W. 1958. Economic Botany of the Cycads.
Economic Botany, 12:3.

Whiting, M. G. 1963. The Toxicity of Cycad. Economic
Botany, 17:271.

Hiroyski, T. 1956. Cycad Poisoning on Island of
Miyako. Okinawa Times, Dec. 13.

Seddon, H. R., Belschner, H. G., King, R. O. C. 1931.
Poisoning of Sheep by the Seeds of Burrawang (Macrozamia
Spirilis). Vet. Res. Report #6, June, Dept. Agr. New
South Wales, Sydney, Australia.

 

Hall, W. T. K. 1956. A Note on Macrozamia and Xanthor-
rhoea Poisoning of Cattle. Australia Vet. J., July :173.

Cecil, R. L., Loeb, R. F. 1955. A Textbook of Medicine.
W. B. Saunders Co. Philadelphia, p. 1459.

Seifter, 3., Dayton, 8., Novic, B., Muntwyler, E. 1950.
The Estimation of Glycogen with the Anthrone Reagent.
Arch. Biochem. 25:191.

Watt, B. K., Merrill, A. L. 1963. Composition of
Foods. Agr. Handbook No. 8. U. S. Dept. Agr.
Washington, D. C.

Cooper, J. M. 1941. Isolation Of A Toxic Principle
from the Seeds of Macrozamia spirilis. J. Proc. Royal
Soc., New South Wales, 74:450.

 

Nishida, K., Kobayashi, A., Nagahama, T. 1955. Cycasin,
A New Toxic lecoside of Cycas Revoluta Thunb. Bull.
Agr. Chem. Soc. Japan, 19:77.

Riggs, N. V. 1956. Glycosylazoxymethane, A Constituent
of the Seeds of Cycas Circinalis L. Chem. and Industry,
35:926.

 

Matsumoto, H., Strong, F. M. 1963. The Occurrance of
of Methylazoxymethanol in Cycas Circinalis L. Arch.
Biochem., 101:299.

 

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

52

Nishida, K., Kobayashi, A., Nagahama, T., Kojima, K.,
Yamane, M. 1956. Cycasin, A New Toxic Glycoside of
Cycas Revoluta Thunb: IV Pharmacological Study of
Cycasin. (in Japanese) Seikagaku, 28:218.

Laqueur, G. L. 1964. Carcinogenic Effects of Cycads
and Cycasin in Rats. Fed. Proc. in the press.

Laqueur, G. L., Mickelsen, O., Whiting, M. G., Kurland,
L. T. 1963. Carcinogenic Properties of Nuts from
Cycas Circinalis L. Indigenous to Guam. J. Natl.
Cancer Inst., 31:919.

The ANRC Reference Chick Diet. 1956. Feed Age, May.

Mantel, N. 1951. Rapid Estimation of Standard Errors
Of Means for Small Samples. The American Statistician,
Oct. :26.

Dixon, W. J., Massey, F. J. 1957. Introduction to
Statistical Analysis. McGrawaHill Book Company, Inc.
New York.

Duncan, D. B. 1955. Multiple Range and Multiple F
Tests. Biometrics, 11:1.

Whiting, M. G. 1963. (personal communication) NIH
Bethesda,.Md.

Popper, H., Schaffner, F. 1957. Liver: Structure and
Function. McGraw—Hill Book Company, Inc. New York,
p. 500.

Encyclopedia Britannica, Vol. 10, 1964. Encyclopedia
Britannica, Inc. William Benton, Publisher, Chicago.

Goldschmidt, S., Vars, H. M., Ravdin, I. S. 1939. The
Influence of the Food-stuffs upon the Susceptibility Of
the Liver to Injury by Chloroform and the PrObable
Mechanism of Their Action. J. Clin. Invest., 18:277.

Gyorgy, P., Seifter, J., Tomarelli, R. M., Goldblatt,

H. 1946. Influence of Dietary Factors and Sex on the
Toxicity of Carbon Tetrachloride in Rats. J. Exp. Med.,
83:449.

Nutrient Requirements of Laboratory Animals. National
Academy of Sciences-~Natl. Res. Council. Publication
#990.

27.

28.

Harper, A. E.

1958.

53

Balance and Imbalance of Amino

Acids. Ann. New York Academy Sci., 69:1025.

Farris, E. J.,

Philadelphia,

Griffiths, J. O. 1949. The Rat in
Laboratory Investigation. J. B. Lippincott Co.
Chapter 13.

ADDITIONAL RESEARCH PROJECTS

Washed Cycad Flour

 

Introduction

Our interest in the toxicity of cycad centered around
its potential harm as a food. The quantitation of the daily
consumption of cycad, in normal use is difficult. However,
cycads are known to be of considerable importance as
emergency foods. We became particularly interested in the
cycad prepared for human consumption in areas in which the
plant is indigenous. This cycad has been washed, dried and
milled (see page 14 of manuscript). Washed nut slices were
purchased from local markets in Guam, mailed to us and ground
to flour in our laboratories. In all, 14 shipments were re—
ceived. These were obtained at various times of the year and

represent many locales of Guam.
General Procedures

Our experimental work began in October of 1962 when
30 male and 30 female weanling rats of the Osborne-Mendel
strain were fed diets containing this washed cycad flour.
The animals were divided into 4 groups of 7 or 8 males and

females. Three groups received 1.5, 5, or 10% washed cycad

54

55

flour in a nutritionally adequate commercial chick starter
ration? while the fourth group received basal diet only. All
animals were housed individually-—feed and water were given
ad libitum. Weights were recorded at the beginning of the

experiment, after 5 days, and thereafter at weekly intervals.
Volitional Activity

Before the end of the first month Of feeding, 1
animal of the group receiving 10% washed cycad flour showed
incoordination of the hind limbs. Because of the earlier
suspected role of cycad in the etiology of amyotrophic
lateral sclerosis, the animals' spontaneous activity was
studied. The male animals were subjected to a week Of train-
ing in revolving drum cages. The number of revolutions pro-
duced by the activity Of each rat was recorded every day for
a period of 2 weeks. The average number of revolutions dif-
fered for each group, but the differences were not correlated
with the level of cycad fed. Female animals were not studied
for volitional activity because of a suspected respiratory in-
fection in the laboratory where the activity cages were

located.
Respiratory Infections

After approximately 3 months, the animals lost a

marked amount of body weight and remained at this reduced

 

8Purina Startena.

56

level for 2 months (figure 8). It was suggested that un-
seasonably low out-door temperatures affected the tempera-
ture in the animal laboratory and this in turn affected the
animals. All female animals fed experimental rations lost
significantly more weight during this period of suspected
respiratory epidemic than females fed control diets (P<(0.01).
The differences in male animals were not significant, but the
recovery of lost weight was slowest for those receiving the

10% washed cycad diet.
Reproductive Response

Males and females of the same diet group were placed
together 4 months after the beginning Of the experiment. We
thought that the added stress of pregnancy might precipitate
a toxic reaction to cycad. The animals did not mate although
vaginal smears indicated a normal estrus cycle. To overcome
possible infections which may have prevented conception,
achromycin was fed at the level of 2 mg/gram of diet for 1
week and 3.5 mg/gram of diet for an additional week. There
was no noticeable beneficial effect from the antibiotic
therapy. Subsequently, a 5% supplement of ground liver
powder, fed for 1 week, or a complete substitution of the
grain ration by McCollum'S Lactating diet in the control
group did not cause any appreciable growth response.

In a separate study, it was Observed that animals

grew poorly when fed the commercial chick starter ration,

57

FIGURE 8

GROWTH OF ANIMALS FED WASHED CYCAD FLOUR

 

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59

but growth was normal when the rats were fed any one Of 3
other nutritionally adequate diets. The commercial chick
ration sold during the winter months contained a coccidio—
stat which affects the food intake of rats and undoubtedly
explains the poor growth Of our animals. Thereafter, a
natural grain ration prepared on campus to our specifications9
was used instead of the chick starter.

Approximately 2 weeks after the final change in
ration, the first litter was born to a control animal; there-
fore, conception probably occurred shortly after this animal
was started on McCollum's Lactating diet, i.e., 4 weeks
prior to parturition. Seventy-five to eighty—five percent
of the mated pairs had litters, some born as late as 4
months after the males and females were placed together.
Because of the dietary complications, the results of the re-
productive study were not conclusive.

Mortality of newborn pups was highest in the control
group. Since these litters were born earliest,the poor
nutritional condition of the mother due to the previous feed-
ing regime could possibly explain the poor survival. Those
that did survive to weaning were maintained on the same
ration as their parents. Weekly weights were recorded for 3
weeks post weaning. Their growth was normal.

The adult animals were continued on the study for 23

months. Occasional infections were treated with achromycin.

 

9see footnote 1.

60

Male and female animals fed control ration or 10% washed
cycad rations were again paired, within the same diet group,
14 months after the beginning of the study. Only a small
number of the pairs actually mated. This was probably due to
the onset of menopause which is reported to occur in the rat
at 450 days of age. Animals born at this time are being

kept to 9 months of age. Weights are recorded weekly. The
tissues will be prepared for pathological examination at the

termination of the study.

Tissue Preparation

Animals that died or appeared moribund were
autopsied and the tissue prepared for the pathologist.
Autopsy included removal and preparation, after fixation in
formalin, of the liver, kidneys, adrenals, spleen, pancreas,
complete G.I. tract, urogenital tract, heart, lungs, trachea
including thyroid and parathyroid, esophagus, salivary
glands, brain, spinal cord and sternum. After macroscopic
lesions were recorded, all tissues were sent to the National
Institutes Of Health (NIH) for microscopic examination.
Grossly visible lesions were photographed and arbitrarily
scored as to severity. At 23 months all surviving animals
were autopsied. The pathologist's findings are not yet

completed.

61

Unwashed Cycad Flour

 

Introduction

Extensive work at NIH showed that the cycad nut meat
not exposed to the washing procedure was acutely toxic to
rats when 5% was incorporated into their basal diet. Simi-
lar trials at our laboratory caused death in as short a time
as 4 days. The NIH group was able to produce in different
species of animals by long term feeding trials a very high
incidence of liver and kidney neoplasms. Because of these
dramatic responses unwashed cycad flour has been used in the
majority of our investigations.

Nuts harvested in Guam were mailed to NIH for pro-
cessing (described on page 17 of manuscript). The nuts were
processed under carefully controlled conditions so that
their potential toxicity would not be changed. The processed
material was refrigerated at 50C immediately after delivery
to our laboratory and was kept at this temperature until the
time of feeding.

Stability of Toxic Factor and_Variations
in Biological Response

The unwashed cycad flour was used in 11 short term
bioassays. These studies were designed to determine the
relative toxicity of various lots of unwashed cycad flour

and the changes in toxicity that may result from storing,

62

heating or cooking of the cycad flour. The effect of the
level Of dietary protein or dietary methionine on the bio-
logical response was also studied. The majority of the bio-
assays were of 5 days duration. A total of 485 Sprague-
Dawley and Osborne—Mendel weanling rats were used. Table 5
summarizes this aspect of the cycad investigation. Many of
these assays are reported in the accompanying manuscript.
Comparison of Toxic Response of Cycasin
and Unwashed Cycad Flour

Cycasin (see page 16 of manuscript) is regarded as
the main, if not the sole, toxic component Of the cycad nut
and has been isolated in crystalline form by a group at
Kagoshima University in Japan. Some of this crystalline
material was made available to us for feeding trials. The
primary purpose of these trials was to compare the toxic re—
sponse caused by chronic ingestion Of cycasin and unwashed
cycad flour.

Groups of 20 males and 20 female Sprague—Dawley
weanling rats were fed either control ration, 1.0 or 1.5% un-
washed cycad flour or 400 or 600 ppm cycasin in the natural
grain basal diet. In addition, groups of 10 male animals
were fed 2, 3, or 4% unwashed cycad flour. All were housed
individually. Feed and water were supplied in unlimited
quantity. Body weights were recorded on the first and fifth
day of the study and weekly until its termination. On the

fifth day, those animals fed 2, 3, or 4% unwashed cycad flour

63

 

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67

were autopsied. The livers were fixed and prepared for
pathological examination. Hemoglobin concentrations deter-
mined by the Drabkin method (J. Biol. Chem. 98:719 (1932))
were within the normal range although animals receiving the
highest levels of cycad flour (4%) had the highest average
values. At the same time, electrophoretic analysis of serum
proteins was done in triplicate. Essentially no differences
were observed in the 3 groups. The animals that were not
autopsied were kept on their respective diets. After 2
weeks, the growth depression of animals fed the cycasin
rations indicated the animals would probably not survive for
an extended length of time. In order to aid in their re-
covery, these animals were first fed a control ration for 1
week after which the initial concentrations of cycasin were
reduced from 600 and 400 to 400 and 200 ppm respectively.
The animals were fed the lower levels of cycasin for 8
months. Finally, all animals were fed control ration for 2
months and then autopsied. Animals that died or appeared
moribund during the study were autopsied according to the
procedure described earlier.

Workers at NIH observed edema in the connective
tissue of the digestive organs and the epicardium Of calves
fed cycad. Collagen is an important intercellular con-
stituent of connective tissue and is distinguishable from
most other proteins by its high content of hydroxyproline.

To determine if changes similar to those seen in the calves

68

occurred in the rats, 3-day urine samples were collected and
analyzed for hydroxyproline by the method of Prockop and
Udenfriend3 (Anal. Biochem. 1:228 (1960)). All animals fed
experimental rations excreted amounts of hydroxyproline
comparable to the controls suggesting that collagen metabol-
ism, as measured by this test, was not influenced by the in-

gestion Of cycad or cycasin.
Feeding Pregnant Rats

One member Of our group found, in repeated studies,
that when cycad was fed to pregnant rats the newborn pups
had lesions characteristic Of those caused by the direct in-
gestion of cycad. Also, when the cycad-containing feeds
were ingested by dams after parturition only, the suckling
animals developed these lesions even though they did not, at
any time, have access to their mothers' rations. This work
proved that the toxic factor of cycad was transmitted through
the placenta and mammary gland.

Because the aglycone of cycasin is similar in chemi—
cal structure to several known carcinogens, it may follow a
metabolic pathway common to these carcinogens. Three chemi-
cals were used for comparison with cycasin. They were in—
corporated into the natural grain diets of pregnant rats at
levels slightly higher than cited (J. Path. Bact. L:393),

as being toxic to weanling rats. Pathological lesions in

 

3determination carried out by M. G. Yang, Ph.D.

69

pups exposed to 200 ppm dimethylnitrosamine, diethylnitrosa-
mine, 400 ppm cycasin or 1000 ppm dimethylaminoazobenzene
indicated that all these chemicals were transmitted across
the placenta.

In another study, 25 dams were fed these 4 chemicals
post—partum. Two pups from each litter, that was suckling
the dams, were killed at weekly intervals. These suckling
pups were also affected by the toxicity even though their
exposure was through the mother's milk only. Body Weights
of the pups were recorded at birth and weekly thereafter.
Physical development was compared to the expected progress
for normal pups of corresponding ages. The mortality was
high in those litters wherein their dams were exposed to
levels in excess of 100 ppm dimethylnitrosamine, 150 ppm di-
ethylnitrosamine, 500 ppm dimethylaminoazobenzene or 300
ppm cycasin. Dams fed these levels or above, produced young
with lesions in several body organs. Those that survived to
weaning are being fed a natural grain ration, free of the
chemicals, until death or 6 months of age. The growth of
those that survived the toxicity is comparable to the con-
trols, except for the animals exposed to 100 ppm dimethyl—
nitrosamine. These weighed significantly less at weaning
and throughout life than animals exposed to all other chemi-
cals. It has not yet been ascertained whether the initial
damage progresses or whether the body is able to regenerate

the damaged tissue when the animals are changed to a control

7O

ration post weaning. These 4 chemicals seem to share a
common property in that they are transferred across the
mammary gland in a form which is able to produce lesions
comparable to those seen when the same compounds are given

directly by mouth.

Feeding Weanling Rats

Weanling Sprague-Dawley rats in groups of 8 were fed
diets containing 125 ppm dimethylnitrosamine or diethylnitro-
samine or 1000 ppm dimethylaminoazobenzene. Their response
was compared with controls, and with that Obtained in the
study already described, wherein the animals were fed cycad
or cycasin. One animal from each experimental group was
killed on the first, second and fourth week of the study.

All other animals were kept for 6 months or until death.
Those that died, appeared moribund in the interim, or sur-
vived until the sixth month were autopsied. Microscopically
Visible lesions developed in some animals from all groups fed
experimental rations. A more detailed pathologic report is
to be included in the doctoral dissertation of the patholo-

gist in our group.

71

Cycad Husks

 

Introduction

In Guam, the natives chew the fresh husk of the
cycad as the nuts are harvested from the trees. The husks
are also dried and later chewed by the men as they work.
Preliminary work at NIH indicated that the husks of the
cycad were about 1/5 to 1/6 as toxic as the nut meat. Short
term bioassays were the basis for this conclusion.

Further investigation into the toxicity of cycad
husks began in July 1964 at our laboratory. A trial was
started to compare the effects of chronic feeding of cycad
husks with those obtained in the unwashed cycad flour feed-
ing trial mentioned previously. Rations containing 6, 9, 12,
18 or 24%.husks were prepared. These levels would correspond
to the toxic potency Of l, 1.5, 2, 3 or 4% unwashed cycad
flour assuming the reported toxic level is correct. Casein
was added to make the husk diets isonitrogenous with the
control. The husks were considered to contribute only
negligible amounts of nitrogen to the diets. This study.
originally planned for 1 year was terminated after 9 days
because of high mortality in the experimental animals.

Those living were anemic in appearance and cold. Gross ex-
amination at autopsy suggested gastrointestinal hemorrhage
to be the immediate cause of death. In most animals, there

was accumulation of fluid in the abdominal and thoracic

72

cavities. Liver, kidney and heart were prepared for patho-
logical examination. High hematocrit and hemoglobin values
determined by comparison with a commercial standard4 sug—
gested hemoconcentration. Both the blood plasma and the
urine were markedly yellow. Positive reactions to Ictotest5

indicated bilirubin was present in the urine. Blood smears

were taken for differential count.
Effect of Environmental Temperature

The lowered body temperatures of the rats fed the
husk diets may have caused a peripheral vasoconstriction and
the apparent anemia. There were however, no differences in
hematocrit and hemoglobin values of 5 animals fed cycad
husks at room temperature (77—830F) as compared to animals
fed the same level of husks when the environmental temper—
ature was raised to 88—900F, Both groups of animals had
elevated hematocrit and hemoglobin concentrations when com—

pared to animals fed control rations under both conditions.
Blood Changes

As a follow-up to the previous study, 15 male

Sprague-Dawley rats of various ages (1-6 months) were fed

 

4Hycel Cyanmethemoglobin Standard, Hycel, Inc.

5Ames Laboratories.

73

the diet containing 24% cycad husks. Blood samples were
taken by heart puncture after 0, l, 2, 5, 7 and 9 days of
feeding, Average hemoglobin and hematocrit values were sig-
nificantly higher on the first day of feeding than at 0
time. These values remained elevated to the end of the ex—
periment. Since there was a possibility that the hematocrit
and hemoglobin concentrations observed in this study may
have been influenced by a lowered food consumption after the
first day (0—5 g/rat/day), another study was initiated. Six
rats were fed the 24% husk diet and another 6 pair—fed a
control diet. The results of this study confirmed the find-
ings of the previous trial. The hematocrit and hemoglobin
concentrations obtained during 4 consecutive days of feeding
were markedly elevated for the rats fed the husks but only
slightly increased for the pair—fed controls (figure 9). The
elevated values of the experimental animals suggested hemo—
concentration and/or decreased plasma volume. Attempts to
measure blood plasma volumes of these animals were not
successful.

Cooperative Studies with the Veterinary
Pathology Department

 

 

Swine

One male and 1 female pig weighing approximately 50

lb each, were fed a pig grower ration containing 2% unwashed

74

FIGURE 9

% CHANGE IN HEMOGLOBIN AND HEMATOCRIT FROM 0 TIME
ANIMALS FED 24% CYCAD HUSK DIET

OR CONTROL DIET (PAIR-FED)

75

 

 

 

 

 

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76

cycad flour. Two control animals were fed the grower ration
alone. The 2 groups of pigs were housed in separate indoor
pens at the Veterinary Pathology barns. Feed and water were
provided ad libitum. The experimental animals ate poorly
and gained weight slowly. One animal of each group was
autopsied after 9 weeks, the remaining after 13 weeks. Both
experimental animals were affected by the toxicity of the
flour. Characteristic lesions in the liver, kidney, heart,
pancreas and G.I. tract were Observed. In addition, con—
siderable fluid had accumulated in the abdominal, thoracic
and pericardical cavities.

In another trial, 3 pigs, 2 weeks of age were fed
various levels of cycad husks. Our purpose was to determine
if a species other than the rat was susceptible to the toxi-
city of husks. Previously we had Observed the most dramatic
responses in younger animals so pre-weaned shoats were used
in the trial. All animals were fed normal cow's milk
previous to their receiving experimental rations. Cycad
husks were first given in milk and later in a mash of grain,
molasses and milk. One control animal received milk or mash
without cycad. They ate poorly, developed diarrhea and
vomited after the first day on experiment. One experimental
animal was dead after 3 days; all including the control
animals were dead by the ninth day. Consumption of husks
varied from 3 grams for the animal that died on the third

day to 15 grams for the one that was autopsied on the ninth

77

day. Gross pathological examination suggested that the

animals died of starvation.

Suckling Shoats

 

Because the toxicity of cycad was found to pass
through the mammary gland Of the rat, we became interested
in determining whether other species would respond similarly.
A sow was fed a grain concentrate containing 2% unwashed
cycad flour immediately after farrowing. Her 9 shoats were
nursed for 8 weeks. Care was taken so that the young did
not have access to the mother's feed. At the end of the
suckling period 3 shoats and the sow were autopsied. The
remaining 6 shoats were weaned to a control ration. Three
of these were autopsied at the eighth and the remainder at
the eleventh month. The lesions in the young, autopsied im-
mediately after suckling were more severe than those in the
mother. Animals killed after 8 or 11 months had marked or

complete regeneration of damaged tissue.

Cow and Calf

In a similar study, a calf nursed while its mother
received 0.1 g of cycad/kg of body weight for 10 weeks and
0.2 g/kg for an additional 9 weeks. The cow was fed a 50-50
mixture of cats and corn at the level of l lb/lOO lb body
weight/day. Both animals had free access to pasture grass.

Routine analyses of blood taken from the cow and calf at

78

weekly intervals were carried out in the pathology depart-
ment. Body weights were recorded weekly. Both animals were
sacrificed after 19 weeks. The calf responded as did the
suckling shoats; it suffered more severe tissue damage than
did the cow.

An attempt was made to locate the fraction of cow's
milk which contained the toxic factor resulting from cycad
ingestion. A second cow, that had recently freshened, was
fed a natural grain concentrate and pasture grass. The
grain was supplemented with 0.02 lb Of cycad flour/100 lb
body weight/ day. Her calf nursed for short periods of time
each day during the study. Most of the milk was taken for
rat feeding trials. The calf served as a partial control
for the transfer of the toxicity and kept milk production at
a maximum. It was recognized that the calf might not show
very pronounced lesions due to the fact that it was getting
milk only sporatically. This is in contrast to the previous
experiment where the calf received all of its mother's milk.

Two feeding trials of 8 or 6 weeks duration were
carried out. In the first study, 52 young male rats (32
days Old) were fed control milk or milk diets made of
fractions prepared from the milk obtained from the "treated”
cow. Centrifugation and acidification (and neutralization)
were used to fractionate the_milk. Each fraction was re-
constituted with the missing fractions from control milk.

"Treated” milk was also heated or stored before being fed.

79

All diets were supplemented with Fe, Cu, I and Mn and then

2
homogenized. Daily consumption and weekly weights were re-
corded. One animal from each of the 9 diet groups was
autopsied after the second and sixth week. Blood samples

for routine analyses were taken by heart puncture. Liver,
pancreas, kidney and heart were fixed for pathological ex-
amination.

Sixteen day Old rats were used in the second trial
because Of the observation that young animals were more sus-
ceptible to the toxicity of cycad. The 3 experimental diets
used contained 90% "treated" skim milk or 10 or 20% "treated"
cream in control fractions. These contained an estimated 40
and 60% fat (dry wt basis) respectively (AOAC Eighth Edition,
1955). The responses of animals fed these diets were com-
pared to those of animals fed normal milk containing 10 or
20% cream. Daily milk consumption and weekly weights were
recorded. Two animals from each of the 5 diet groups were
autopsied at 1, 2 and 3 weeks after the beginning of the
study. Blood samples and tissues taken then, and at the
termination of the study at 6 weeks, were sent to the path-
ologist for examination. Both studies failed to establish
with certainty the fraction in which the toxic factor was
located.

The cow and her calf were autopsied after 5 months.
Neither animal had pathological lesions at the time of

autopsy.

80

Horses

Four horses (2—3 years old) were used in another ex-
periment. Since neurological distrubances have been reported
as a cardinal manifestation of cycad ingestion, it appeared
desirable to check this species. Horses were used as the
experimental animals because they are reported to be particu-
larly susceptible to neurological disturbances.

Two of the horses were fed unwashed cycad flour as
2% of their daily food intake of cats (1/2 lb/100 lb body
weight) for 15 weeks and as 4% for an additional 9 weeks.

The other animals received oats without cycad. Routine blood
analyses were performed each week. Body weights were re—
corded at the same time. Serum glutamic—oxalacetic trans-
aminase and serum glutamic-pyruvic transaminase were fol-
lowed by the pathologist. One experimental and 1 control
animal were autopsied after 24 weeks. A neuro-pathologist
reported that the brain and spinal cord of both animals were
normal. Lesions in the liver, pancreas, heart and kidney
were characteristic Of lesions seen in other species fed
cycad. The remaining horses have been fed pasture grass,
hay and oats free of cycad for an additional 9 months. The
animals appear to be in good health. There are no clinical

signs of any neurological disturbances.

MICHIGAN STATE UNIVERSITY LI

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