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ABSTRACT

ACUTE SELENIUM TOXICOSIS IN THE BABY PIG

by Robert Max Diener

Sodium selenite was fed to 3sweek-old.pigs to determine the symp-
toms, hematology and gross and microscopic lesions. One lot was fed a
basal ration while in 5 additional lots the basal ration was supple-
mented with 0.5, 1.0, 2.0, h.0, and Boo mg. selenium.per lb. body weight,
respectively. The two higher levels were lethal in approximately h8
hours, while the lower dosages were progressively less toxic.

Signs of anorexia, vomiting, diarrhea, hypothermia, dyspnea, and
incoordination were noted. Hemoconcentration was present in the acutely
affected animals while serum.glutamic oxalacetic transaminase values were
increased.approximately 10 to 20 fold.

The most severe lesions were noted in the groups fed the largest
amounts of selenium, Gross tissue changes included: fatty livers, con-
gested blood vessels; congestive catarrhal gastroenteritis, often with
gastric ecchymoses and ulcers; occasional epicardial hemorrhages; and
cyanosis.

Histopathological manifestations were most severe in the liver, kid-
neys, and gastrointestinal tract. Fatty metamorphosis, centrolobular ne-
crosis, and congestion were noted in the liver. Degeneration and fatty
changes of the renal tubules plus varying degrees of congestion were
present in the kidneys. The most evident gastrointestinal lesions in-
cluded.ulceration, exfoliation, and.erosion of mucosal epithelium and

congestion, hemorrhages and leukocytic infiltration.

ACUTE SEIENIUM TOXICOSIS IN THE BABY PIG

Robert Mex Diener

A THESIS

Submitted to
the College of veterinary Medicine
Michigan State University of
Agriculture and.Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Veterinary Pathology

1961

6,»

.734) / r: I]
.I’// 7/59’

ACKNOWLEDGEMENT

The author is deeply indebted to Dr. C. K; Whitehair of the Depart-
ment of veterinary Pathology for his continual guidance and encouragement
during this research; to Dre. C. C. Morrill and R. F. Langham of the De-
partment of veterinary Pathology for their suggestions and assistance
with the histopathologic aspects of this research; and to Dr. G. H.
Conner, Department of Surgery and Medicine, for his aid and constructive
criticisms.

In addition the invaluable help of Dr. Howard Stowe, in conducting
the experimental.work, and Miss Nancy Malik, in.preparing the tissues for
examination, is sincerely appreciated.

Last but not least the author wishes to express his gratitude to his

wife, Deborah.Ann, for her unflagging loyalty and help in the preparation
of this thesis.

ii

TABLE OF CONTENTS

I MRODUCT ION O O O O O O O O O O O O O O

REVIEWOFLITERATUREoooo......

A.
B.

c,

Acute Selenosis . . . . . . . .

Chronic Selenosis . . . . . . .

Distribution of Selenium in the

D. Factors Preventing Toxicity . .

MATERIAISANDMETHODS ........

RESULTS
A.
B.

C.

GeneralResults . . . . . . . .

Sigs O O O O O O O O O O O O O

Patholog...........

Liver
Kidney . . . . . . . .
Gastrointestinal Tract
Heart and Lungs . . .

Miscellaneous Changes

DISCUSS ION O O O O O O O O O O O O O 0

SUNNARYANDCOMIUSIONS . . . . . . . .

REFERENCESCITED............

iii

0 O O O

Tissues

\ONWWU

12
15
15
17
19
19
21+
27
29
51
35
59

LIST OF TABLES

Table Page

I Body Weight Changes, Survival Times and Blood Chemical
Findings in Pigs Fed Various Amounts of Selenium . . . . 16

II Signs and lesions Resulting from Selenium Ingestion by
P138 0 I O O O O O O O O O O O O O O O O O O O O I I O O 18

iv

LIST OF FIGURES

Figure Page

1. Comparison between a pig fed selenium for 12 days
and a control animal . . . . . . . . . . . . . . . . . . 2O

2. Attempted bile duct formation at the periphery of
Of a liver lObule O O O O O O O O O O O O O O O O O O O 22

3. Centrolobular destruction and large basophilic
regenerating hepatic cells in the liver . . . . . . . . 23

h. Renal degeneration with fatty metamorphosis and
necrosis . . . . . . . . . . . . . . . . . . . . . . . . 25

5.. Degeneration of gastric glands . . . . . . . . . . . . . . . 28

6. Denuding of the epithelium from the ileum.. . . . . . . . . . 5O

INTRODUCTION

The role of selenium in animal and human health has become of major
importance in recent years. It has been known for many years to be a
toxic substance and recent research has demonstrated it to be a required
nutrient for most species of livestock. In addition, contamination of
foodstuffs such as cereals is of public health concern.

The toxicity of selenium has long been a problem which, despite much
research, has defied complete solution. The effects of various toxic
doses on laboratory animals has been recorded by many workers. Reports
on toxic levels for farm animals are less numerous. Selenosis in sheep,
horses, cattle, and swine has been studied but detailed histopathological
findings were often limited or lacking entirely.

Recently reports have been published demonstrating that selenium is
a growth factor for most species of livestock. In New Zealand, the ad-
ministration of selenium to lambs suffering from "ill thrift" reduced
mortality and improved gains (Drake gt_al., 1960) while Jelly (1960) re-
ported improved weight gains in calves fed rations supplemented with se-
lenium. It has been shown to reduce the incidence of muscular dystrophy
in lambs when fed to pregnant ewes (Muth gt_al., 1959). Subcutaneous in-
Jections have also proved beneficial in reducing the incidence of muscu—
lar dystrophy in lambs (Lagace, 1961).

The public health aspects of selenium poisoning should not be mini-
mized. Smith and co-workers (1956) stated that bad teeth, skin disturb-
ances, chronic arthritis, and cardio-renal problems were found in 111
families residing on seleniferous Dakota soils. ’Ill health was also

prevalent in workers engaged in the processing of selenium, according to

Dudley (1956a).

The recent findings that selenium is a nutritional as well as a
toxic factor emphasizes the need for additional research, especially on

the histopathologic aspects of acute toxicosis.

IJTERATURE'REVIEW

The literature on selenium is so voluminous that only the references
pertinent to its toxicity and role in nutrition in animals is reviewed in
detail.

According to Moxon and Rhian (19h3) the first authentic record of
selenium poisoning ("alkali disease") in livestock.was written in 1856
by Madison, an army surgeon stationed at Fort Randall, South Dakota. He
reported a fatal disease of cavalry horses. The roles of geological for-
mations, vegetation, and weather in selenium toxicity were extensively
reviewed by Trelease and Beath (19h9).

Selenium toxicosis has been divided into acute and chronic types.
Highly toxic or lethal doses produce the acute type, while small repeated

doses over long periods of time produce chronic symptoms.

Acute Selenosis

 

The minimum lethal dose of selenium for acute toxicosis in the common
laboratory animals has been discussed by many researchers and much dis-
agreement exists. Frankeiand.Moxon (1936) at the South Dakota Experiment
Station reported that intraperitoneal injections of 3.25 to 3.5 mg. of
selenite per Kg. of body weight or 5.25 to 5.75 mg. selenate per Kg. body
weight was lethal to rats in two days. Smith and co-workers (191.0) at
the National Institutes of Health stated that the method of administration
did not affect the toxicity of selenium. The minimum lethal dose for rats,
rabbits, and cats was given as 1.5 to 3.0 mg. per Kg. body weight. This
dosage was confirmed on dogs by Anderson and beon (19h2). However, or-
ganic selenium compounds were less toxic than the inorganic, regardless

of the route of administration (beon 33 51., 1938).

Lethal doses in farm animals have also been studied, although on a
limited scale. Miller and.Williams (19h0a) found the minimum lethal
dose of sodium selenite to be 1.5 mg. per 1b. body weight in horses, h.5
to 5.0 mg. per lb. in cattle, and 6 to 8 mg. per lb. in swine. Khttler
g§_§l. (1961) reported that 80 mg. of sodium selenite per 100 lbs. body
weight were lethal and ho mg. per 100 lbs. body weight were toxic to
sheep.

Among laboratory animals the signs of acute toxicosis are essen-
tially identical. Czapek and.Weil, Modica, Jenes, Smith, Anderson and
Moxon are cited by Trelease and Beath (l9h9) as having described the
following: garlicky breath; signs of nervousness, fear and excitation;
vomiting and diarrhea; dyspnea followed by opisthotonos and tetanic and
clonic muscle spasms; and finally somnolence and death.

Acute signs of toxicosis in South Dakota farm animals were de-
scribed by Beath and associates (1935b). Manifestations included a
characteristic "tucked.up" stance with abdominal pain and bloating;
diarrhea; elevated temperatures; rapid, weak.pulse; labored breathing
and.pale or cyanotic mucous membranes.. Prostration occurred shortly
before death .

Descriptions of the gross and microscopic lesions of acute sele-
nium poisoning are extremely limited. Smith (1937a) noted that cats
‘which died in 7 to 11 days from selenium poisoning exhibited fatty de-
generation and centrolobular congestion of the liver and fatty degenera-
tion of kidney tubules. Focal hemorrhages and edema of the lungs were
the only other lesions observed. Gross and microscopic lesions in 10
sheep were recorded by Rosenfeld and Beath (19h6a). These included

hemorrhage and congestion of the endocardium, lungs, spleen, kidneys,

liver, gall bladder, pancreas, intestines and omasum. Necrosis and de-
generative changes were present in the liver, kidney tubules and gastro-
intestinal tract. iMuscle atrophy was also noted. Miller and Williams

(l9h0a)_made almost identical observations in a pig.

Chronic Selenosis

 

Levels of selenium which produce chronic toxicosis vary widely
with diet and species. Doses of 10 ppm to 52 ppm in the diet of rats
have been reported by Smith (1959) and Frankeand Potter (1935) to pro-
duce chronic manifestations. Smith (l9h0) reported that 1h ppm sele-
nium in the diet of rabbits produced only moderate liver fibrosis, while
3 to 13 ppm were lethal to a dog after 135 days (Rhian and Moxon, l9h3).
In pigs 2h.5 to 392 ppm of sodium selenite in the feed produced death
in 10 to 99 days (Miller and Schoening, 1938). Rosenfeld and Beath
(19h5) at the Wyoming Experiment Station agreed with Smith (l9h0) that
1.0 to 1.5 mg. of inorganic selenium per Kg. per body weight per day
was necessary to produce important pathological changes.

Toxic effects of selenium are not restricted to the host animal.
MCConnell (l9h8) reported that in rats 2.5 to 9.3% of the selenium is
transferred to the young via the mammary glands and that the selenium
found in the milk is entirely in the protein fraction as part of an
organic complex. Trelease and Beath (l9h9) cited the work of Franks
and associates (1936) in which as little as 0.01 mg. of selenium in-
Jected into incubating henS' eggs caused deformities in the chick. Yet,
in spite of this extreme sensitivity of the chick embryo, young and
adult chickens are apparently unaffected by toxic grains. ‘Westfall 33
233 (1938) demonstrated that selenium is transmitted through the‘pla-

cents to the fetus in rats and cats, but no deformities were noted. He

concluded that the extraordinary susceptibility of the chick embryo to
selenium is a species characteristic not shared by mammals. However,
more recent work by Wahlstrom and Olson (1959) indicated that sows on
a ration containing 10 ppm selenium gave birth to an increased number
of dead.pigs. In addition, conception rate was lowered and the number
of services per conception increased. The number of days from weaning
to estrus was also increased.

The principle signs of chronic poisoning in laboratory animals are
reduced appetite, stunted growth, emaciation, and loss of hair (Trelease
and Beath, 191.9). Lesions are mainly restricted to the liver. Lillie
and Smith (19h0) reported that cirrhotic changes were first noted after
5 weeks in rats on'a seleniferous diet. After 11 to 15 weeks, increas-
ing fibrosis replaced the proliferative changes in the liver, while the
kidneys and other organs remained essentially normal. Hepatic cell ade-
nomas and low grade hepatic cell carcinomas are apparently associated
'with the cirrhotic changes in the livers of these animals, according to
Nelson _e__t_ _a_1_._. (1915). Of 53 rats fed 5 to 10 ppm selenide for 18 months,
ll.had neoplastic livers, while in a corresponding control group of 350
rats only h tumors appeared. Seifter gt_al, (l9h6) reported the occur-
rence of thyroid adenomas in rats fed selenium.

In farm animals selenosis is of two types: a fairly acute, often
fatal type called "blind staggers" and a milder, more chronic form
called."alkali disease." The signs and lesions of the two syndromes
are different (Draize and Beath, .1955). Signs of blind staggers in-
clude: aimless wandering, anorexia, impaired vision, and finally ab-
dominal pain, dyspnea and death (Beath at 31:, 1931.). Alkali disease,

on the other hand, is characterized by emaciation, loss of hair,

deformation and sloughing of hoofs, and general loss of vitality
(Trelease and Beath, 19h9). Signs may be delayed for several months
(Beath, 1935a).

The lesions of both syndromes have been well documented. There is
essential agreement that the morbid changes in blind staggers includes
smooth muscle atrophy of the gastrointestinal tract, gall bladder and
urinary bladder; congestion of liver and kidneys; areas of focal necro-
sis in the liver; mucosal degeneration of the stomach and small intes-
tines; and hemorrhages of'the epicardium. The lesions of alkali disease
are more chronic. Atrophy of the heart, atrophy and cirrhosis of the
liver, degeneration of the kidneys, and erosions of the Joints are most
often noted while petechial hemorrhages of the epicardium an; degenera-
tive changes in the gastrointestinal tract are uncommon (Draize and
Beath, 1935; Miller and Schoening, 1938; Miller and.Williams, l9h0b;

Moxon, 1937; Rosenfeld and Beath, 19h6a; and Schoening, 1936).

Distribution of Selenium in the Tissues

 

 

The distribution of selenium in the tissues of the horse, sheep,
hog and calf was studied by Dudley (1936b). He reported that in
acutely poisoned animals, the liver, kidney, and spleen contained the
greatest quantity (h.0 to 25.0 ppm, wet weight) of selenium. Work on
dogs by Rhian and beon (19h3) confirmed Dudley's results. McConnell
(l9hl) round that the liver and kidneys of rats contained the highest
levels of selenium two hours after injection of radioselenium.

In chronic toxicosis the distribution of selenium in the body tis-
sues differs somewhat from acute cases. Dudley (1936b) reported that
in chronic cases the concentration of selenium was much higher in the

kidney than in the liver, while in acute cases the reverse was true.

Kidneys analyzed by Kuttler 33.5%: (1961) contained at least ten times
more selenium than the livers, thus further substantiating the results
of Dudley. Rosenfeld and Beath (l9h5) found the largest amounts of se-
lenium in the livers and kidneys of sheep, while the brains and muscles
contained only minute amounts. Fat was negative for selenium.

Amounts of selenium in the blood vary widely. In acute cases
Dudley (1936b) found the concentration to range between 7 and 27 ppm,
‘whereas in chronic cases the range was 0.2 to 5 ppm. Most of the sele-
nium was present in erythrocytes as part of a protein complex. The
serum, plasma and fibrin were negative for selenium. This was generally
substantiated in cats by Smith et al. (1937b). McConnell (19hl) re-
ported the maximum.concentration in the blood 15 minutes after one sub-
cutaneous injection of sodium selenate in rats. The selenium at first
~appearedmore abundantly in the plasma than in the erythrocytes, but
this was reversed after 6 hours, apparently because selenium was elimi-
nated from the plasma at a faster rate than it was incorporated into
the red cells. Minyard and co-workers (1957) at the South Dakota Ex-
periment Station determined selenium blood values in a large number of
steers and concluded that values of more than 2 ppm indicate probable
tissue damage; 1 to 2 ppm, possible damage; 0 to 1 ppm, no tissue dam-
age. Blood levels dropped rapidly when selenium was discontinued.

Hematological Changes are not restricted to the transportation of
selenium, however. .Anderson and Moxon (l9h2) reported.marked increases
of hemoglobin and.packed corpuscle volume in dogs injected subcutaneously
'with sodium selenite. Blood phosphorus, non-protein nitrogen, calcium,
ascorbic acid, and sugar were decreased. Franke and Potter (1935) noted

hemoglobin increases up to 25 gm. per 100 ml. blood during the first 9

to 17 days of selenium ingestion in rats. After this time, anemia de-
veloped. In-vitro studies by Rosenfeld and Beath (l9h8) demonstrated
that plasma had the'ability to reduce selenate to selenite plus a vola-
tile selenium, whereas in erythrocytes no such reduction took place.
However, most of the conversion from the selenate to the selenite took
place in the liver and the author concluded that toxic reactions are
due to the selenite, and that transformation followed this pattern:
selenate to selenite to "combined form" to volatile and elemental sele-
nium, .

Excretion studies have somewhat clarified the metabolism of sele-
nium in the body. McConnell (191+2) reported that in rats 5 to 10% of
the original subcutaneous dose of selenium was excreted through the
lungs in 2h hours. Gortner and Lewis (1939) and.Anderson and Moxon
(19hl) indicated that 20 to 50% of the ingested selenium is excreted in
the feces, a major part of this being unabsorbed selenium. The kidneys
excreted 30% of the absorbed selenium, according to work in sheep by
Rosenfeld and Beath (l9h6a). Furthermore, the same authors (l9h5)
found that it takes approximately 60 days for sheep to deplete their
tissues of selenium, while cats and rats require only two weeks (Smith
SE 2&3: 1937b; Anderson and Moxon, 19hl). only small amounts of sele-
nium are found in the urine shortly before death, due mainly to the kid-
ney damage which prevents excretion and results in larger tissue stor-

age (Miller and Williams, 191+0b; Rosenfeld and Beath, 1915).

Factors Preventinngoxicity
A variety of rations and.supplements have been fed to animals in
an effort to prevent or reverse selenium toxicosis. Franke and Potter

(1935) demonstrated that alternate feeding of seleniferous corn and

10

rations devoid of selenium gave growth and food consumption curves of
rhythmic decreases and increases in rats. Diets high in protein decreased
the toxicity of selenium in rats (Smith, 1939; Gortner, 19h0; Lewis et_
51., 19h0). This was confirmed in sheep by Rosenfeld and Bcath (l9h6b).
Fels and Cheldelin (19148) noted that the inhibition of yeast growth by
selenate was reversed by the addition of methionine. However, Klug SE
Elf (1952) reported that 0.5 to 1% methionine increased weight gain only
slightly in rats on a 23 ppm selenium diet, while it did not result in
lower selenium content of the livers. He therefore refuted the idea
that methionine antagonized or inactivated selenium in 3.1.19. . Olson ‘33.
9.}: (1958) stated that methionine had its greatest effect at the lower
selenium levels. Choline and betaine also gave some protection.

Attempts to cure selenosis by various chemicals have also been
made. Moxon gt_al. (19h0) reported that p-bromobenzene increased the
‘excretion of selenium in dogs and steers. This could not be duplicated
by Westfall and Smith (19A1) in rabbits. The recovery rate of steers
poisoned with selenium was not increased by the administration of p-
bromobenzene (Minyard gt_al,, 1957). Rosenfeld and Beath (l9h7) re-
ported that the toxicity of selenium in rats was not altered by the
ingestion of increased amounts of ascorbic acid while injections of
potassium iodide increased the adverse effects of selenium.

Arsenicals have been used repeatedly for prevention of selenium
toxicosis. Rhian and Moxon (l9h3) reported that 5 Ppm sodium arsenite
prevented weight loss, anorexia, and anemia in dogs, but did not result
in lower selenium content of tissues. The theory that arsenic inhibits
absorption of selenium could not be confirmed in further work (Moxon st

51., 19h5). Organic arsenicals, viz. 0.01% arsenilic acid and.0.005%

11

3-nitro-h-hydroxypheny1arsonic acid, prevented symptoms and increased
the gains in growing pigs fed 7 to 11 ppm sodium selenite in the diet
(Wahlstrom gt 21., 1956)' The protective effect is not as pronounced
in steers, according to Minyard and associates (1957).

In summary, it is evident from the literature that much more de-
tailed information is needed on the effects of ingestion of selenium
by animals. According to Trelease and Beath (19h9) many unexplained
factors enter into the selenium problem. Accurate minimum dosages of
the various forms of selenium for each species of animal are still un-
known. No investigator has yet reported on the cause of the final col-
lapse of animals. Likewise the matter of individual tolerances to se-

lenium has not been fully explained.

MATERIALS & METHODS

General Procedures. A litter of 12 Yorkshire pigs was used to

 

study the effects of feeding various levels of sodium selenite. The
litter was farrowed on April 6, 1961. Each animal was injected intra-
muscularly with 2 ml. iron dextran (Haver-Lockhart) when one week old
to prevent anemia. The litter was weaned 0n.April 23, 17 days after
farrowing.

A period of acclimation to the experimental environment and ration
was instituted after weaning. The animals were placed in individual
21- by 36-inch galvanized.metal metabolism cages equipped with wire
floors and individual feed and water crocks to facilitate accurate meas-
urement of feed and excreta.

On May 1, the animals were divided into six lots of two animals
each. Each lot contained a male and a female of approximately equal
weight. The control group consisted of two males. Lots 1 through 5‘
‘were fed sodium selenite at levels of 0.5, 1.0, 2.0, h.0 and 8w0 mg./1b.
body weight, respectively, and Lot 6, which served as controls, was fed
only the basal ration.

Sodium selenite was added to the milk of each animal once daily un-
til death or termination of the experiment. Appropriate amounts of se-
lenite were pipetted from an aqueous solution containing 10 mg. (Baker's
grade) sodium selenite (Na28e03) per ml. In pigs with decreased appe-
tites the amount of milk used to dilute the selenium was reduced to in-
sure complete ingestion of the selenite. The animals were weighed and
their feed crocks washed immediately before each daily feeding of sele-

niunu - .

l2

15

Ration. Three times daily all animals were fed the basal diet which
consisted of 250 m1. homogenized.whole milk and free choice of a starter
mash. The starter contained the following ingredients: ground corn,
69.5%; soybean oil meal, 28.0%; limestone, 1.00%; trace mineral salt
(high in zinc), 0.50%; dicalcium phosphate,0.80%; and a vitamin-antibio-
tic supplement, 0.25%. The supplement included: Bacitracin, h.0 Gms./
1b.; arsanilic acid, 3.96%; vitamins: A, 600,000 units/1b.; D2, 500,000
units/1b.; E, hoo I.u./lb.; Riboflavin, 800 mg./lb.; Niacin, u,ooo mg./
1b.; 1312, no mg./1b.; choline chloride, 20,000 mg./lb.; Pantothenic
acid, 1,600 mg./1b.; and trace minerals. Fresh water was always avail-
able.

Signs and Hematological Examinations. Signs were recorded at fre-

 

quent intervals. Daily temperatures were included.

Blood samples from the anterior vena cava were collected just be-
fore the experiment started and four times during the course of the ex-
periment. leukocyte counts, hemoglobin, packed cell volume (PCV), serum
glutamic oxalacetic transaminase (SGO-T) and serum glutamic pyruvic
transaminase (SGP-T) determinations were made and recorded. SGO-T and
SGP-T activity was determined by the method of the Sigma Research Labs
oratories (1957). The van den Bergh test for total serum bilirubin was
determined on pig E5932 11 days after the experiment started.

Post-mortem Techniques. Necropsy examinations were performed on
all animals as soon after death as possible. Animals which could not
be examined immediately after death.were stored in a refrigerator. Con-
trol animals, as well as those that were in moribund condition, were
killed by electrocution (120-v A.C.). Tissues for microscopic examina-
tion included: liver, gall bladder, kidney, urinary bladder, spleen,

esophagus, stomach, small intestine, colon, reproductive tract, thyroid,

1h

adrenals, pancreas, lung, heart, aorta, hoof, tail, proximal part of
tibia and skeletal muscle (cross section of semimembranosds mm.).

All tissues except striated muscle were fixed for 2% to 36 hours
in Zenker's solution (modified by the exclusion of acetic acid). fter
fixation, tissues were washed for 2h hours and then stored in 80% ethyl
alcohol. Muscle sections were fixed and stored in 10% acetate-buffered
formalin. Bone was decalcified with formic acid and sodium citrate.

Sections were cut at 6 microns and stained with Harris' hema-
toxylin and eosin. In addition, formalin sections of heart, liver, and
kidney were stained for fat with Sudan Iv. All histopathological pro-

cedures used are described in the Manual of Histologic and Special

 

Staining Technics of the Armed Forces Institute of Pathology, Washington,

D.C. (1957).

RESUETS

A summary of the general results given in Table I indicates that
the toxicosis produced was fairly well correlated with the amount of se-
lenium fed.

Survival times exhibited marked individual variations, expecially
those fed the lower dosages of selenium. The two pigs in Lot 5 died in
N6 and 50 hours, respectively. The pigs in Lot h survived only 5h hours.
Ihilot 3, one pig died after 3 days, the other after 5 days. Lots 1 and
2, however, were extremely variable. In Lot 2 one pig died after 60
hours while the other survived for 13 days; in Lot 1 only one animal
died of selenium poisoning. The other was still alive at the end of the
experiment and was placed on a selenium-free diet for 6 days to see if
recovery would result.) During this time the animal regained its appe-
tite and increased 1.25 lbs. in weight.

ReSults from a limited number of hematological tests indicated that,
of the examinations performed, only hemoglobin, PCV, and SGOJT values
were altered. Samples of blood from pigs E5938 and.E59h0 taken shortly
before death indicated a marked increase in hemoglobin and percentage
of packed cells. The increase was approximately two-fold over identical
tests performed prior to the start of the experiment 3 days previously.
However, pigs in Lots 1 and 2 exhibited only moderate increases by the
10th day of the experiment.

SGO-T determinations were also highly elevated. Values from 335
to 870 SigmaéFrankel (S-F) units were obtained from the animals tested.
Samples collected on these same animals before feeding selenium ranged

between 21 and 32 S-F units. 0

15

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17

Other tests performed included total leukocyte counts, differential
leukocyte counts, SGPHT, and the van den Bergh test. The results of
these determinations were not significantly different from normal values.

alarm

Signs and gross lesions observed are summarized in Table II.

In general, the severity of the observed signs varied directly with
the dosage of selenium fed. Loss of weight was a pronounced and consist-
ent manifestation in all selenium-fed animals. From 2.5 to 3h% of the
initial body weight was lost by the treated pigs while the two control
animals gained A9 and 59%, respectively (Table I). The most consistent
weight loss occurred in the two lots fed the highest amounts of selenium.
In less than 3 days these animals lost an average of 17.7% of their body
weight. Results were more variable with lower dosages of selenium. Pig
E5933, which was fed 0.5 mg. selenite per pound body weight for 12 days
made steady weight gains immediately after removal of the selenium from
the diet.

Moderate to complete anorexia was noted in all animals. Almost com-
plete anorexia occurred within 2h hours in the three lots fed the highest
amounts of selenium. In the two remaining lots, anorexia was delayed sev-
eral days and was less severe.

Vomiting and diarrhea were pronounced, especially in Lots h and 5.
In these lots, diarrhea started approximately 2h hours after the initial
ingestion of selenium and continued for 6 to 8 hours. The animals ap-
peared.uneasy and in acute distress. Their backs were arched and heads
lowered. An initial period of severe vomiting was followed by varying
periods of retching and diarrhea. The stool was white and pasty in ap-
pearance and became progressively more fluid during the course of the

experiment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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18

19

These signs were followed by a period of progressive apathy and
somnolescence and culminated in coma and death. Gastrointestinal symp-
toms were milder in Lots 2 and 3, while Lot 1 was not visibly affected.
Deaths in Lots 2 and 3 were delayed.

Cyanosis in varying degrees was manifested_in all the treated pigs.
The snouts, ears, tails, and hoofs were most severely affected, being
cold and bluish in appearance. A sharp horizontal line of demarcation
separated the dark, purple, distal half of the hoof wall from the more
normal appearing proximal half. This phenomenon was noted on all treated
animals and was thought to be due to severe congestion and stasis of
blood in the large veins of the laminar corium, although this could not
be substantiated histologically.

A pronounced dr0p in body temperatures occurred 12 to 28 hours be-
fore death in 8 of the 9 pigs killed with selenium. The body tempera-
tures of the h pigs in Lots h and 5 averaged 93 F during this time while
the range in the less acutely ill animals was 97 to 100.8 F. The low-
est reading obtained from the control animals was 101.2 F.

Other signs included dyspnea, which was quite prominent in the
most severe cases, and occasional nervous manifestations. The latter
were restricted mostly to incoordination and usually occurred before the
animal entered the comatose stage. However, two of the animals (E5938
and.E593h) suffered from a slight chores of the head and cervical area.

No tetanic of clonic spasms were noted.

Pathology
Liver. The most prominent single gross lesion observed in this study
‘was a pale, yellow-brown, rough, friable liver. There was littIe varia-

tion in color. However, the liver in the very acute cases was more

20

 

Fig. 1. Comparison of control pig E5930 (above) , and pig E5933 from
Lot 1 after 12 days (below).

21

yellow and "fatty" whereas in the less acute cases the texture was more
granular and crumbly. Pig E5933 was the exception. This animal was fed
a selenium-free ration for 6 days and at the termination of the experi-
ment the gross appearance of the liver was normal. ‘

Microscopically the liver lesions observed can be divided into an
acute group where congestion and centrolobular degeneration prevailed,
and a more chronically affected group displaying early regenerative ac-
tivity.. Allelivers except those from the controls manifested fatty
changes.

The acutely 111 group included seven pigs, all of which died during
the, first six days. Congestion and acute centrolobular degeneration were
especially conspicuous in this group. The congestion was most pronounced
in the livers from animals in Lots h and 5, although marked individual
variation existed. Areas near the capsule. were most severely affected.
Here the central veins and surrounding sinusoids were packed with
erythrocytes. The congestion was not always restricted to the center of
the lobule, however. In various sections portal veins were filled with
blood and the areas around them contained small amounts of serofibrinous
exudate and escaping erythrocytes .

Degenerative changes were more severe in the centrolobular. areas.
Cloudy swelling was widespread and affected a majority of the central
parenchymal cells. More advanced changes were evidenced by numerous
pyknotic and karyorrhectic/nuclei which were not always restricted to
the central portion of the lobule. Increased numbers of Kupffer cells
were present in most sections. .

Fatty metamorphosis in the liver was widespread and severe.. Only

in the liver of pig E593h, which succumbed after ingesting 25.9 mg. of

 

Fig. 2. Attempted bile duct formation at the periphery of a lobule
in the liver from pig E5935. H81E stain; X1175.

25

 

38E stain;

Centrolobular destruction and large basophilic regenerating

hepatic cells (H) in the liver from pig E5935.

x5 70 c

Fig. 3.

21+

selenium, was the fat restricted to scattered individual liver cells.

. In the rest of the pigs, a few too many fat globules were present in the
cytoplasm of albiost. every parenchymal cell which was not in an advanced
stage of degeneration. Therefore, under low-power magnification, a
lobule stained with Sudan IV appeared as a red, granular disc encircled
by a narrow rim of periportal connective tissue and containing a central
vein surrounded by a zone of degenerating cells. Accumulation of fat
was generally more pronounced in the peripheral third of the lobule.

In addition, the size of the intracellular fat globules were much
smaller in the acutely 111 animals, whereas in the more chronic cases
these globules tended to coalesce until the nucleus was displaced to
the border of the cell. However, no "lipodiastaemate" (a large multi-
cellular Structure containing a huge central fat droplet) as described
by Hartroft (1950) were found.

In the three animals which survived more than .10 days, very early
regenerative changes were noted. These included slight increases in
collagen and early fibroblast proliferation in periportal areas, appar-
ent attempts at bile duct proliferation (Figure 2), and the appearance
of large, often multinuclear, cells (Figure 3) which are associated with
regeneration (Schiff, 1956; Beamsand King,‘19l+2).

In the all bladder the most'consistent findings were varying de-
grees of mucous degeneration and desquamation of the mucosal epithelium.
m. Gross lesions in the kidneys were not observed. Ureters and
urethra were apparently normal. The urinary bladders were in various
stages of contraction and no gross changes were evident.

. Histopathological lesions were present in the kidneys of all the

selenium-fed pigs and included: congestion, usually in'the medulla;

25

 

'Fig. h. Renal degeneration.(E5936) with fatty metamorphosis of the
proximal convoluted tubules (F), and necrosis of the ascend-
ing thick segment of Henle's loop. H&E,stain; X830.

26

cloudy swelling and coagulation necrosis of the proximal convoluted tub-
ules; and various degrees of tubular fatty metamorphosis. No distinct
relationship could be found between any of the above processes and the
amount of selenium fed, although congestive changes were most severe in
the acutely ill cases.

Generalized congestion .of blood vessels was a predominant feature
in the kidneys of, the animals that died during the first three days.
The degree of congestion was most severe in the tissues from pigs in
Lot 5. In the kidneys of these animals the glomerular capillaries. were
distended and engorged with erythrocytes. The interlobar, and arcuate
veins, (as well as the capillaries of the medulla, were engorged with
erythrocytes. Perivascular edema and occasional interstitial hemor-
rhages were also noted. In the bladder, moderate congestion of sub-
epithelial capillaries and edema of the muscularis mucosa prevailed.-

Degenerative changes varied widely in the kidneys. In the acute
cases, the predominant change was cloudy swelling, which was most con-
(spicuous in the loops of Henle. The most severe degenerative changes
generally occurred in the ascending thick segments of the loop. In
these medullary segments pyknotic nuclei and granular, poorly staining
cytoplasm were‘especially noticeable (Figure 1+).

Fatty metamorphosis, which. was very pronounced. in all but two of
the kidneys, affected the convoluted and terminal portions of the proxi-
mal tubules almcst exclusively (Figure 1+). Ascending segments, distal
. convoluted tubules, and collecting tubules were almost devoid of fat.

The urinary bladderwas remarkably free of lesions. Only mild
degrees of congestion, edema, and occasional areas of desduamation of .

transitional epithelium were noted.

27

Gastrointestinal Tract. At necropsy, the stomachs of the acutely ill

 

animals contained varied quantities of mucus and undigested feed. The
mucosa appeared slightly hyperemic and in the fundus small circumscribed
brown clots of blood were noted. These areas were present in over half
of the animal .

The intestines appeared distended with gas, dark red, and highly
congested in the acutely affected animals but more nearly normal in the
chronically affected cases. The lumina were usually empty and only in
the terminal portions of the colon was fecal matter found. The mesenteric
vessels were engorged and dark blue in color. Eesenteric lymph nodes
were not unduly enlarged and the esophagus and pancreas appeared grossly
normal. No ascitic fluid was noted.

Microscopically the stomach and small intestines exhibited the
more marked lesions. Congestion, hemorrhage, leukocyte infiltrations
and erosion or deSQuamation of the epithelial lining were most often
found.

Lesions in the stomach were quite similar in all cases. The most
severe manifestations generally occurred in the animals fed the largest
' aunts of selenium, while the pig which had been on a selenium-free
diet for 6 days exhibited no obvious lesions.

Congestion in the stomach appeared most severe in the lamina
propria. Capillaries were engorged with erythrocytes and often so dis-
tended that the whole area appeared hemorrhagic. The mucosal layer was
not the only on- affected, however. The larger vessels in the subm -
cosa and even in the mesenteries were not infrequently plugged with
cells, and thrombi were seen in various vessele. In these areas of
severe venous stasis, c .;a often caused some mild separation of con-

nective tissue fibers. This was most prevalent in the submucosa.

28

Degeneration of gast;.: glands in pig 35956.

of ves.

's

~ . J

and thrombus (T). HdE stain; X128.

 

Note congestion

29

U
(D
('0.

(D

:1

(D

H

ation of the gastric epithelium was common in most of the
pigs. The de res and extent of damage varied from isolated areas of
mild surface erosions to complete denudation and mucous degeneration of

large numbers of gastric glands, leaving only the connective tissue

skeleton of the lamina prop ia (Figure 5). Hemorria e and coagulation

O
H
O

aseation necrosis were usually evident in these areas and the coagu-
lated blood pigments near the surface of the lesion were a vivid yellow-
brown.. Generally, however, the lesions were not so severe and consisted
mostly of mucous degeneration of the surface cells and neck cells, with
some sloughing of the former. Distention or the capillaries and mild
infiltration of lymphocytes and plasma cells in the lamina propria com-
pleted the usual picture.

The lesions found in the small intestine were usually very similar
to those found in the stomach. Congestion of blood vessels was pro-
nounced in almost all cases and leukocyte infiltrations of the lamina
propria were much more conspicuous than in the stomac . Peyer's patches
were conga ted and hemorrhagic in the acutely affected animals (Figure

/\ 1 -- . - -- -
o; and areas of hemorrhage into the lumen OI the intestine were repeat-

Denuding of the epithelium of the villi was another common finding
in acute toxicosis (Figure 6). In the more chronically affected animals,
however, the damage was usually restricted to mucous degeneration and
scattered coagulation necrosis with isolated erosions.

Lesions in tre colon were relatively mild. They consisted of con-
gestion and a small amount of epithelial erosion. Only pig ES9hl demon-

V

4. a. " lug—hr , J -
seraeed narked ca ene ation.

s. Crossly the heart aid lungs exhibited few lesions. No

abnormalities of the lungs were observed, while the heart lesions were

 

 

Fig. 6. enuding of the epithelium from the ileum of p;; 359hl. Note
the hemorrhage and congestion in the lamina propria and Peyer's
patch. HdE stain; X150. .

51

usually restricted to epicardial petechial hemorrh: as present in the
region of the coronary groove. Pig 35952 was the only animal demonstrat-
ing any abno-lal amount of pericardial fluid. About 5 cc. of clear fluid

was seen in this case. Endocardial hemorrhages were absent. Fluid was

- ~. - v“ .- ‘» ° I A. 1 -‘~~—- 11‘ 4-
never osserved in the thoracic cavi cy in denon straoie amounts at autopsy.

[.1

Histoj ashological changes in the hear were restricted to capi lary
congestion, a few myocardial and epicardial hemorrhages and a moderate
“mount 0: fatty metamorphosis. The hemorrha- as were usually localized
in the neriva scular fat of the coronary vessels and were probably due to
diapedesis. Evidence of fatty etauorphosis could not be detected in the
sections stained with hematoxylin and eosin, but the sections stained
with Su Jn IV reveale d numerous small to medium fat sp herules dispersed
throughout the myocardial muscle fibers. Thelsp she rules often appeared
in small clustersa ound the nuclei of muscle fibers but otherwise were

appare ent ly evenly distributed throughout the myocardium. All pigs ex-
cept the control animals were affected. More advanced degenerative
changes could not be demonstrated.

Various degrees of capillary congestion were the only persistent

CFC-“ZQQ ’\

c- round in the lungs. Mild interstitial thickening was noted in

T~seallemons Ti<s m~e Changes. Gross lesions other than those which have

 

already been des ”lb d were not evident. Lo en -gement or significant

abnormality of the lymph nodes or splenic tissue was n ted.

)

Histopathological changes were noted in the adrenal s and and lymph

0"“

nodes. In the adrenal gland the medulla usually congested with

€.Jthrocvtes. Occasionally the zona glonerulosa and zona reticulata

6

were similarly .ggected. The lymph nodw generally dam nstrated mild

'1? “""'""V fl :1”
ca anus o: subc ape

L.

lar con gestion, hemorrhage, and edena. This was more

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UiSCLSSION

o ‘ n~ ...L- <0" 4-~‘--- -" ~~ .— ‘ 1;- ‘s‘ ’3' .9 f' ‘
The results oi b--S study confirm ~d he Iindinfs oi Liller and

Williams (1940a) in the" the single oral lethal dose of selenium (in

H .9 -‘ fl v ‘ - a —- r‘ ‘- . 'I- “- - fivfi v- - Q 5" "
the form 0. soaiun salon-.e in SL1“: is apprOXi_ateiJ c mg. per pound
‘- f‘ . '01- -'- I -'— J— ;- ‘ )1; ' ‘ “A "'3‘: 1 't- 3; . _ \A " ‘3 ,8
of bony weighs. at ni s level deutn resulted in approxila -ly 4 hours.

433 3393+ and 33937 demonstrate the individual variaoiiisy t? at ex-
isted. The :ormer ingested a tOt al of only 23.9 mg. of s- -lenite, yet
died aitcr c3 hours, wnile the latter consumed 93.8 mg. and lived for
120 hours. gurthermore, the report of Fitzhu ugh et al. (l9#u) that fe-
males are more susceptible to seleniu mtoxic ty thanr £3168 could not be
verified in the small numoer of pigs in tnis experiment. On the con-
trary, the two animals which survived for the longest period of time
were both females.

igi- o anorexia,v omition, dia arrhea, hypoth ermie, dyspnea, and
incoordination were note d, and paralleled the observations reported by
heath (iyjh) and Miller and Williams (l9hOa). However, no corneal

rcling, or "pus hing"

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was observed. All the signs seen,
except the chorea, were directly prOportional to the amount of selenium
administered. Garlicky breath which was noted by several early workers
was not detected in these pigs.

laboratory findings indicated that severe hemoconcentration oc-

curred in the very acutely affected animals, while pronounced cellular

necrosis was omewhat more delayed. Hemoglobin values from pigs E5938
and 33¢£O, taken shortly before death, had increased by h3%. nderson

and lozon (19L2) reported similarr res ults in dogs.
he most sensitive index of cellular daraoe, h wever, ap peared to

““0

be the SGO-T lev-ls. The SGO-T determination is used to measure an

(D
L)

‘zyme present in tissues normally, but which has been released to the

" . n a ‘ O43 ‘ . ‘ .- . '
blood stream as a result 01 b rea Md .In 0. the cell carrier by ne iOSis.

Cornelius t al. (l939) reported the born: for pigs to be 22 .7: 5. h
S—F units. This corresponds well with t‘ nge of 21 to £9 8-? units
in the 12 pigs before the Start of this experiment. lt was further re-

- 4" fl '0' ‘ J -u ~-'~ ,‘V’t- ‘,-V 3’) . . '-.-A : . to A71: -‘ - ‘- ’4' s _ 1 -:
pcruea that this tees was or value in the diapnosis o- wnite muscle dis-

495-2360 S-. units '“ affected animals hile normal calve s and lambs
anged between l9 and 99 S-F units. - gace (l96l) ootained somew what
higher results. It was of interest, therefore, to find that, in the
more chroni ally poisone ani 'mals of t? is experiment, a ra.ge of 535

to 870 S-F units we obtained af er 9 days on a seleniura diet. Two

U)

determinations taken from pigs in Lots h and 5 showed only a moderate
rise (9 and 170 S-F units).

It is not known exactly which tissues caused the arxed elevation
c: bee—T values. According to Cornelius (l939) only six organs contain

signiiicant amour sof glutamic- oxalac e ic tr

f)
:3
(I)

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K")
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in the pig.

re, in descending order: heart, skeletal muscle kidney, liver,

\o

pancreas, and spleen. Of these, the heart, muscle, and kidneys contain
r re than twice the amoun of er.zyme than the est of the organs. Yet,
this is not where the most severe histo: athological lesions were found.
It may be the alterations in cellular permeabi lit ty occur without the

obvious his tolegical signs of cell necrosis.

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counts, were new Signiiicantly altered. SuP-T Valu~s were never ele-
V("-;-- ": o I r ‘I-" F‘ 1

Vateu \ln lac. they appeared d

no a ’21-» - o ~
epresseu due p-osaslyt che iact c

the pig contains significant amounts of this enzgre only ' the hi dney.

The single van den Bergh test for total bi Vb; n, which was run on the
blood from pi 23932, wa within the normal iange and thus-in harmony

with the report of Smith at al. (1940) that no bili"uoincaia was ever
ound in chr nically pois ned rats and cats.

" - H. A " N ‘1 A ‘ IL —.- .a A Wfi-I q-‘A-‘fil‘fl ‘
Gross lesions OOsefch at necropsy ger .er all y pa-al_eled those seen

1 “_ _- fl _’_ .1- —v"’ - fl ‘ ’3 _;___-~_“ A‘ ~._~_\_; A,“ r"‘ a a. _- fi 4‘“.
by uralze and leash (193)) in sneep and cat is -"iected by blind stag-

- H ' 4 ‘ ' . w, 4. ‘ ° .1. .- ' -.- :J .'.‘,.- -, .-‘- _ v.-.
gers and will net be enumerated in detail. h wevcr, the-c are some
‘ O a, 4-. V .0 ' v n J. 1 3 I _‘ I .‘ v- . 4“ J"l"‘._ H ‘ ‘ --, ‘
QlfierenCcS which warrant furtner discusSion. he is the large degree

0: fat present in the livers of the pigs fed selenium in this experiment.
Ho menti n of fatty livers was made by Draize, whozierely described th's
cigan as acutely congested and containing areas of focal necrosis.
Rosenfeld L i heath (l9L 6a), on the other har nd, made not e of the fact
that the livers from sheep which died of acute rang intoxication and.
blind staggers were usually soft, yellow, and contained extensive fatty
changes and areas of necrosis.

Further disparities in the gross lesions were restricted chiefly
gree of the hemorrhagic manifestation. None of -We pigs in this
experiment exhibited the -etechial or eccth tic hemorrhages in the lungs,
endocardium, and peritoneun that were reported by Rosenfeld and Beath
(1926a) for sheep. Chang s in the colon were also not as sever and

could be la1eled as congestive, ra her than hemorrhagic enteritis.

Histcpathological changes were most se‘ere in the liver, kidneys,

and gastrointestinal tract. In general the lesiont found were quite
similar to those reported by Miller and Williams (l940a) in one pig

nilled by 10.3 mg. sodium sel nite per pound body weight. However, the

”"3 "‘1"“' V 1 ‘
—gcneratlve muscle changes in the

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p 2-; 7— in the lungs, were not observed.

r. vs '1 - - q -\ -I q N - “
n sure;ary of aicroscopic l've" lesions lnC'dQSQ congestive changes,
- _' ‘a— -4- A A - .1 . fiA ~A‘ qu.r-.-.—-. 4“». ‘_ w. on 4- m
various degrees of Centroiooular degeneiaoion, and severe iatty metamor-
.. _ -0- " .L. - -‘ '. l M- . a..- -- 1- 4..
phos‘s. C“ these, the fatty cnang-s pro'aciy repiesen-oa t- earliest
,—,. - 4.. 4.: ... -- 1 - ‘| w . 77 . -03.. " "."fi ‘ «our,
2a ifestations oi liver e age. iartro o (-9po) demonst:ated the t tr .ese
changes take place within 2% hours in rats on a csoline—aef cient ration.
- A '0 IL— » ‘v- upfiuL-«n nua- “‘On
It was uncreiore not surpriSing b0 find narhed lousy alterations in the
-: ~c ,1”. ‘ a: a .-_«.v.__-,, a“ ‘8 I“ c
p is.) “I l&&cn Lieu. bu- vw- 0;..‘f ‘7 Aourh) O
"— ‘ "J" 'L"/‘- : N. “x A 4“ ‘0‘ a. ~~ Ah ‘fi 1 ‘ -: ~ A
As flfbb b“: lat is apparently present as snall globules which have

a tendency to coalesce within the cell as time goes on, until finally
the sphiiale oecorzes so large as to ‘isplace the nucleus to the periphery

n the liver sections from.

P0

. -\ t1. -. ’r\‘35 l‘ -. I . ," v—.—‘- 1'
‘ll. This p..enornenon was quite aaraed

Q I ‘ 4- ("s '1 .0 ‘a‘ro I ‘0‘.’ ‘-f‘ f‘ W
this exec hient, and the more chroni cal_y into.icated anilals displayed

P 1 - A.“ ”a. A‘h 3" 4-. -a - ‘A " .. -: .“r‘
lion larger globules on the average than the more a utely ll pigs.

however, this process aacarently was not oi sufficient duration to pro-
described by: {artr oft in the rat in which
se'eral 0: these distended cells rupt cure with the f rnati on of an enor-

mous m‘ ticellular structure. It was also observed that the fat—contain-

0

ing cells w-re nore concentrated at the peripheral half of the lobules
than in the central half, whereas in choline-deficient rats the affected
celii appeared more in the midzonal and central areas. Furthermore, al-
most co plete rev reel of fatty met- orphosis was achieved in rats by
discontinuation of the choline- de fici ent diet for 5 to h d ys. Yet, no
microscopic evidence of diminished fat accumulation was noted in pig
25933 after 6 days on a normal, selenium- -f;c- diet, although the gross

rancc of the liver was essent ‘ially norma Apparently th e hepat o-

*

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cellular daia;- produced by selenium is of a more severe nature than that

6

produced oy a cholinesdeficient diet and is not - ily reversed.

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cit;-a;fl, O. G., Ielscn, n. A., an; ~iiss, c. i.: ins cronic Oral
rp +— -.-‘ - - ‘ ,v,— d .-.‘ ~-.. -2 '2 *2: I - r‘ r“ ‘
_oxici y of Seleiiug. J. Pneriicoi. ail iggtl. ;ierap., Co, (1944
F. f‘
”A film
297“C>‘0
-11 - "f vr -, 4. T 7:. r. v- ;-, .--- -. ....- A ,, - .7 _ . .
Fraile, a. u., —:d Pot er, J. A.: n ”eJ -oIichu- cccarring Later-
‘ . _'.. n,--.--.A - .1! ’1". ' *3 ,T_ I;..._‘:'-..'\‘ - ~ .. ‘| Al":
89.1.1.7 J.-. DC:— uwln 8:432;- .2183 OJ. P;.L~n-C 2 COM... vvu... :2 s U - -.'--u: 0 J LO ’ (153/ ):
f" If f‘:
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Mllu ,Ars

. - v, 2‘ .- ~ 'r
enic aid Vanadium. U.

94.-“ ...: .—1:;o {2-13:3 $58, (1922,63 : LE‘L} _L§9.
'34 - 32 5- A-Z Chronic Seleniux Poisoninv of D"s
.\ h I“ ‘ . v I A ‘- ‘ r’ ."
b4 gloo;-j :ro;ein. J. hucr,, 19’ (1990/: 103- id.

Gortner, n. A., and Lewis, H. 3.: The Retettion and Excre
Selenium after the nosinlstra*io. Sodiu u. Sele
J. Pharmacol. andE tl. Therap., 67, (1939): 558-56h.

ha-troft, W. S.: ‘ccumulation of Fat in Lever Cells and
aste;:ata Pre eding Xteriment Dietary Cirrhosis. Anat.
(;;,o): 61-87.

.mento
eJ Ze ala and

the Effect of

A Preliminary Ti eri
e f nVet. J., 8, (

Calves.

Klug, H. L., Harshfield, R. 3., Pengra, R. M., and I.oxon
acthioaine ad Selenium Toxicity. J. Kutr., #8, (1952):

“uttic-, n. L., Liarble, D. W., ano Blincoe, C.: Scrum a
ncsidics T llowing Se lenium Injections in Sheep. Ar. J.

422-L'r28.

Legace, A.: Effect of
Jo-Ao‘vlol'ivo’ 158) (1961):

lenium on White Muscle Disease
188-190.

Lewis, E B., Schultz, J., and Gortner, R. 5.. s;etary Pratein
and the Toxicity of ma Seler -ite in the White Rat. J. Iharracol.
and Expt . Theiap., 68, (l9h0): 292-299.

Lillie, R. D., and Smith, M. 1.: His to nes of Hepatic Cirr4031s
in Cnronic Fo od Mlen sis. Am. J. Path., l6, (1920): 223-228.

vr

.,
,4 o
AX. . o

LcConnell, Distribution an Excretion Studies in
after a Sitgle Subtoxic Succutaneous Injection of Sodiu;

J. 3;-3mac 01 0

tion of

ite to White Rats.

in Lipodi-

Selenium
1900): 13.

,A.L..
#09- #20.

t-e Pat
3' -lte
1+2P{-L+37 o

tcConnell, K. P.: Respiratory Em retion cf SelCLium Sosdied'vish
RéiiOuCElvo Isotope. J. Biol. Chem., 1&5, (1942): 55-60.
McCo-nell, K. P.: Passage of Selenium Thrc-;- the - macry Glands
of the Shite Rat and the Dl--leuEiCZ of SElLHQLfi in the Milk Pro-
tCiJS After Subcutaneous Inuectio; of Sodiam Selenate. J. Biol.
Chem., 173, {19A8); 655-657.

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