THE TOXEGSTY 0F ARSIIMUC mm 1% SWiNE
AND RATS AS AFFECHZ} BY WATER ENTAKE AND
PRGTEER LEVEL

Thesis for the Degree of M. S
MICHIGAN STATE UNEVERSITY
Mamon W. Vorhies
1967

ABSTRACT
THE TOXICITY OF ARSANILIC ACID IN
SWINE AND RATS AS AFFECTED BY
WATER INTAKE AND PROTEIN LEVEL

by Mahlon W. Vorhies

Reduction in water consumption and variation in the quantity of

dietary protein were evaluated as factors affecting the toxicity of

arsanilic acid for rats and swine.
In rats, arsanilic acid toxicosis was produced with a ration con—

taining 24% protein and 0.5% arsanilic acid. A 2/3 reduction of water

decreased the length of time for toxicosis to appear. Lesions included

gastric ulcers and cerebral hyperemia and hemorrhages. The rats fed

an 8% protein diet and 0.5% arsanilic acid and control rats had no

apparent lesions.
Pigs given 44 mg. or 64 mg. of arsanilic acid orally daily for
8 days by a dose syringe and with limited water available developed

signs and lesions of arsenic toxicosis. The clinical signs were pos-

terior paralysis and blindness.. The cerebrum, especially in the tem-

poral region dorsal to the optic chiasma, had lesions of hyperemia,

hemorrhages, neuronal degeneration with satellitosis and neuronophagia,

and perivascular accumulation of lymphocytes and neuroglial cells. The

lumbar area of the spinal cord was hyperemic with hemorrhage and gliosis.
Also noticed was endothelial damage of small vessels in the dorsal horn

of the gray matter, with perivascular accumulation of lymphocytes and

Mahlon W. Vorhies

neuroglial cells. The peripheral nerve sectioned with the spinal cord

had accumulations of lymphocytes.

The pigs fed rations containing 0.02% arsanilic acid with levels
of 8% or 19% protein and given.water varying quantitatively from 120 ml.
daily to free choice (average, 1644 ml. daily) had no signs or lesions
of arsenic toxicosis.

Analysis for arsenic trioxides (A5203) stored in the liver averaged
5 p.p.m. in pigs with clinical signs and lesions of arsenic toxicosis,
3.3 p.p.m. in pigs with 0.02% arsanilic acid in the diet and 240 ml.
water daily and 1.80 p.p.m. in the control pigs fed 0.02% arsanilic
acid and water ad,libitum..

It was concluded that both the quantity of water and the level of

protein may be factors in arsenic toxicity. The results also confirm

previous reports of the safety of arsanilic acid when used as

recommended.

 

THE TOXICITY OF ARSANILIC ACID IN
SWINE AND RATS.AS AFFECTED BY

WATER INTAKE AND PROTEIN LEVEL

BY

Mahlon W. Vorhies

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1967

 

ACKNOWLEDGEMENTS

The author wishes to express his appreciation and thanks to his
major professor, Dr. Stuart D. Sleight, for his guidance and encourage—
ment during the course of the investigation and the writing of this
thesis. The author also wishes-to thank Drs. Stuart D. Sleight and
C. K. Whitehair for their assistance in acquiring financial aid for
this project.

Also, the author wishes to thank Drs. D. J. Ellis, C. C. Morrill,
and R. F. Langham for serving on his guidance committee and for giving
constructive criticisms of this manuscript.

A great deal of appreciation must be expressed to Dr. Edward

Bicknell for his encouragement and valuable advice.

To

My wife and daughter

iii

INTRODUCTION. . . . .

REVIEW OF LITERATURE.

MATERIALS AND METHODS

RESULTS 0 O 0 O 0 O 0

DISCUSSION. . . . . .

S UMPIARY O O O O O O 0

REFERENCES. . . . . .

VITA. O O O O O O O 0

TABLE OF

iv

CONTENTS

Page

Table

LIST OF TABLES
Page

Summary of experimental procedure and groups of animals
given arsanilic acid (AA). . . . . . . . . . . . . . . . 11

Summary of weight changes, mortality, and feed and water
intake of rats in Experiments 1 and 2. . . . . . . . . . 15

Summary of weight gains, liver arsenic trioxide content,
and water and feed intake of swine in Experiments 4
and 5 O O O O O O O O O O I O O O O O O O O O O O O O O O 2]-

Figure

LIST OF FIGURES

.Page

Temporal region of cerebrum dorsal to the optic chiasma.
Satellitosis with neuronal degeneration. Pig Experi—
ment 3, Group 1. Hematoxylin and eosin. x 187.5. . . . . 18

Higher magnification of Figure 1. Notice neuroglial

cells, lymphocytes, and neuronophagia. Hematoxylin and
eOSi-n. x 469. O O O I O O O O O O O I O O O 0‘ O O O O O O 18

Dorsal horn of lumbar spinal cord. Perivascular accumu—
lation of lymphocytes and neuroglial cells (A). Satel—
litosis and neuronal degeneration (B). Pig Experiment 3,
Group 1. Hematoxylin and eosin. x 187.5. . . . . . . . 19

Higher magnification of Figure 3, Part A. Notice neuron—
al degeneration, lymphocytes and neuroglial cells. Hema—
toxylin and eosin. x 469. . . . . . . . . . . . . . 19

Higher magnification of Figure 3, Part B. Dorsal horn

of lumbar spinal cord. Perivascular accumulation of
lymphocytes and neuroglial cells. Hematoxylin and

eosin. x 469. . . . . . . . . . . . . . . . . . . 20

vi

INTRODUCTION

Arsenic is an important toxic heavy metal as well as a growth
and appetite stimulant depending upon the amount of arsenic given. The
toxicity may be peracute to accumulatively chronic, depending upon the
amount, type and rate of arsenic absorption and excretion. Starting
in the early 1950's many commercial swine and poultry rations contained
organic arsenicals to stimulate faster growth. A commonly used organic
arsenical in baby pig starters and growers is arsanilic acid. When
organic arsenicals have been used as directed by manufacturers, no authen-
ticated cases of toxicosis have been reported.

A syndrome in young pigs, which appears to be related to organic
arsenicals because of history and clinical signs, has been seen by the
author and other veterinarians in the Midwest. This syndrome was
noticed in young pigs during the winter months when water intake was
very low. If, in fact, this condition was an arsenic toxicosis, the
mechanism was thought to be the accumulation of organic arsenicals due
to decreased arsenic excretion in the urine.

The effect of total water deprivation in swine was measured by using
a pig weighing 9 Kg. at the beginning of the experiment. By the end
of 6 days without water the pig had lost 1.8 Kg.; hemoglobin had in-
creased from 9.0 to 10.8 Gm./100 ml. and packed cell volume from 30.5
to 36.5%, respectively. The pig became gaunt and weak. It had been

given 340 Gm. of feed daily; on the 4th day only 300 Gm. were eaten;

2
food was refused on the last 2 days. The pig chewed and salivated a
great deal, apparently in an attempt to produce fluids for the dry oral
and pharyngeal areas.
The purpose of this project was to determine whether water depri-
vation and/or low protein levels might be contributing factors in the

toxicity of organic arsenicals.

REVIEW OF LITERATURE

Ehrlich, in 1907, developed arsphenamine as a medicant for
syphilis. This provided the foundation for the use of organic arseni-
cals as chemotherapeutic agents (Frost ggual., 1955). The beneficial
effect of feeding inorganic arsenic was first described by Gies in
1877. The therapeutic use of arsenic as a stimulant was described a
century earlier by Fowler, and its empirical use goes back to the time
of Galen in 200 A.D. (Frost g£_a1,, 1955).

The growth—stimulating effect of organic arsenicals on poultry and
swine was first reported in the late 1940's. Morehouse and Mayfield
(1946) mentioned the growth-promoting effect of 3 nitro- and 4 nitro-4-
hydroxy phenyl arsonic acid. Carpenter (1951) conducted the first
reported tests of the growth-stimulating property of 3 nitro-4-hydroxy
phenyl arsonic acid in swine. Evidence of toxicosis occurred with
high levels, but the arsenical caused the pigs to have smoother hair
coats and a thriftier appearance than controls. Carpenter and Lars.n
(1952) described the control of swine dysentery with 3 nitro-4-
hydroxy phenyl arsonic acid in experimental herds. Frost and Spruth
(1952) reported favorable results in an experiment with swine which
were fed 0.01% to 0.02% arsanilic acid in the diet until reaching market
weight. There was no toxicosis from arsanilic acid at levels of 0.01%
to 0.02% of the diet. Several organic arsenicals have since been

tested for growth stimulation and swine dysentery prevention (Eagle and

4
Doak, 1951; Frost, 1952, 1953, 1961, 1963; Frost, Overby and Spruth,
1955; Hanson 25 31., 1955; Smith, 1961).

Hogan and Eagle (1944) discussed the toxicity, effectiveness and
variations among organic arsenic compounds on a pharmacologic basis.
They stated that the toxicity was related to the degree of arsenic bound
to the tissues. The most significant property related to systemic
toxicosis was the amount of arsenic bound in the erythrocytes. The
nontoxic compounds were not bound to tissues and were rapidly excreted.

Eagle and Doak (1951) discussed the biologic activity of arseno—
benzenes in relation to their structure. The phenyl—ring compounds
with acid radicals were less toxic but also less effective than the
parent phenyl arsonic compound. Amine-substituted groups reduced the
toxicity but not the effectiveness of the parent phenyl arsonic acid,
but arsanilic acid was nearly as effective and less toxic (Jones, 1957).
The effectiveness of organic arsenicals was thought by Ehrlich in 1907
to be dependent upon the rate of arsenic liberation from the benzene
ring as arsenic trioxide.. This rate was thought to be affected by
substitutes on the benzene ring (Eagle and Doak, 1951). The bacteri-
cidal action was thought not to occur until arsenic trioxide was libera—
ted from the ring and attached to the sulfhydryl enzyme systems of
microorganisms.

It was reported that animals that consumed arsenic—containing
tissues from other species did not absorb the arsenic (Coulson, Remington
and Lynch, 1939; Overby and Frost, 1962). The location of arsenic
storage in swine, following oral administration of organic arsenicals,
was reported by Scheidy (1953). The greatest arsenic accumulation was

in the liver and kidney. Katsura (1957) reported that when arsenic

5
was fed to young dogs for a 1- to 2—month period it caused 10 to 20
times normal levels of arsanic.in visceral organs. When the length of
time was increased to 4 months, an increase of only 2 to 4 times normal
occurred in visceral organs, except the kidneys. The arsenic content
in skeletal and cutaneous structures increased during the long-term
administration. Katsura (1958) interpreted these results to mean that
low-level administration of arsenic over long periods resulted in inhi—
bition of arsenic absorption from the gastrointestinal tract. Subse-
quently, the arsenic stored in the organs migrated to the bone and
hair. The levels of arsenic in the kidneys remained high because of
continuous excretion of arsenic in the renal filtrate.

Overby and Frost (1960), in studies of excretion of arsanilic acid
in swine, reported that more.arsenic was excreted in the feces than in
the urine. At the end of a lO-day feeding trial, the intake and ex-
cretion of the arsenic was in approximate balance. Withdrawal from
the arsenic diet caused a rapid excretion in urine.

Vallee, Ulmer and Wacker (1960) reported on the pharmacologic and
biochemical aspects of arsenic. They stated that arsenic was toxic
only in the trivalent state. The reduction of pentavalent compounds
and partial oxidation of the.arsenobenzene groups was required for
metabolic effectiveness ig;gi££9_or 13:32223 Organic pentavalent
compounds penetrated tissues rapidly but were considered to be physio—
logically inactive. Although most of these compounds were excreted
unchanged, small amounts were reduced, allowing trivalent arsenoxides
to reach the cells. The metabolic effectiveness of organic arsenicals

was explained by this reasoning.

6

The enzyme systems, in which arsenic was involved, were listed by
Vallee 2; a1. (1960). They were pyruvate oxidase, lactic dehydrogenase,
alpha glycerophosphate, cytochrome oxidase, and the sulfhydryl enzymes.
The element phosphorus can be replaced by arsenic in many enzymatic
reactions. The example listed was the phosphoglyceraldehyde dehy-
drogenase system. Arsenic can also replace the nitrogen in choline.

Peoples (1963) stated that the work of Vallee _£__1, (1960) was
excellent but that the.conclusions were derived mostly from work in
rats. He stated that arsenic metabolism in rats was unique and that
these animals are unsuitable.as a test animal for research on the
toxicity of arsenic. Peoples gave arsenic trioxide orally to cows to
determine whether residues.wou1d be in tissues or milk. Arsenic was
not detected in blood or milk, and there was a lack of storage in
tissues. Excretion in urine nearly balanced the daily intake. Rats
fed similar diet stored quantities in the erythrocytes as well as the
visceral organs. Some preliminary studies by Peoples indicated that
tissue-bound arsenic was.entire1y pentavalent and there was no evidence
that it is partially reduced to the trivalent forms in tissues. These
differences in metabolism make it necessary to reconsider present con—
cepts of arsenic compounds.as.cumulative poisons.

Overby and Fredrickson.(1963) studied the mechanism of the effect—
iveness of organic arsenicals.. They showed that organic arsenicals
were not degraded to inorganic arsenic or reduced to arsenoxides, as
had been reiterated for years.. They fed radioactive arsanilic acid to
chickens and conducted chemical analyses of tissues and excretions;

14 74

they reported that the.ratio.of C to As in a double-labeled mole-

cule was unchanged and that the radioactive arsanilic acid was excreted

7
unchanged. No oxidized or reduced metabolic products of arsanilic acid
were found in tissues. These.resu1ts tend to disprove that the effective—
ness of arsenic occurs only after degeneration or reduction to arsenic
trioxides.

The toxic level of arsanilic acid in swine rations, as determined
by Notzold g£_§l, (1956), was 0.04% in the total diet. The maximum
recommended feeding level was 0.01%, but 0.02% was well tolerated.

Some hypercalcification was reported in swine fed 0.02% arsanilic acid
for 2.5 months, but no clinical signs were reported (Frost and Spruth,
1956).

Factors reported that could alter the toxic levels of arsanilic
acid were phosphorus (Grimminger, 1962), ascorbic acid and para-
aminobenzoic acid (Vallee gt_a1,, 1960), and selenium (Olson §t_§1,,
1963).

An example of arsenic toxicosis related to decreased water intake
was a heavy mortality which occurred in dehydrated lambs following
parenteral injection of diethylamine acetarsol (Staples, Salisbury and
Van Der Wonder, 1958). When.1ambs were given therapeutic intramuscular
injections of diethylamine acetarsol (20 mg. arsenic equivalent) many
deaths occurred. In experimental studies, lambs were deprived of water
for 5 days and were then injected with arsenic. Five of the 6 lambs
died in 8 hours.

An example of arsenic toxicosis related to decreased excretion of
water was described by Jansch.and Brachetka (1939). Chickens with gout
retained 6 times the amount of arsenic oxide in their tissues as did

normal birds when injected with atoxyl (arsanilic acid).

8

Clinical signs of arsenic.toxicosis have been reported (Goodman and
Gilman, 1956; Thienes and-Haley, 1957; Oliver and Roe, 1957; Garner,
1961; Smith, Perdue and Kolar, 1962). These signs varied as to dose
and whether the compound was organic or inorganic. In peracute cases
no clinical signs were usually noticed -- just death. In acute poi-
soning, clinical signs included salivation, vomition, and hemorrhagic
diarrhea with mucous shreds...Sudden deaths occurred after exhaustion
and collapse. In chronic or-accumulative toxicosis, some of the signs
mentioned were albuminuria,.hematuria, motor paralysis, tremors, joint
pain, pronounced edema, hyperkeratosis, dermatitides, and blood dys-
crasias. Organic compounds.with phenyl rings caused similar clinical
signs, but many signs were-related to disturbances in the central ner-
vous system. Blindness,.convulsions, tremors, staggering, goose-
stepping gait, and paralysis.were reported.

The lesions related to the.toxicity of arsenic were described by
Jubb and Kennedy (1963),.Thienes.and Haley (1957), and Goodman and
Gilman (1956). The major lesions were related to the cardiovascular
system. The histologic lesions were hyperemia, hemorrhage, and edema.
Capillary permeability was increased and endothelial necrosis was
present.

In the gastrointestinal tract the irritating effects of arsenic
produced loss of the mucosal.epithelium, ulceration, edema, and necrosis.
The kidneys had congestion of the glomeruli accompanied by hemorrhage
and loss of plasma protein. In some of the tubules there were albuminous
casts. Tubular necrosis occurred as the arsenic became more concen-
trated in the collecting tubules. Cloudy swelling, fatty degeneration

and centrolobular hepatocellular necrosis were histologic lesions in

9
the liver. When repeated exposure to high levels of arsenic occurred,
atrophy of the liver with jaundice was present. The results of hemato—
logic examinations during toxicosis showed aplastic anemia and agranulo—
cytosis (Thienes and Haley,.l956). The vascularity of the bone marrow
was increased, but cell production centers were destroyed.

Hurst (1959) and.Jubb.(l953) reported that central nervous system
lesions were prominent in organic arsenical toxicoses in monkeys and
swine. Lesions reported were hemorrhagic encephalitis, with edema and
focal areas of necrosis.. Polyneuritis of peripheral and optic nerves
was a prominent lesion.. In severe cases, neurons in the spinal cord
were necrotic. The cerebral lesions were mainly vascular in origin and
occurred in both the white.and-gray matter. The lesions were typically
reported as multiple focal.hemorrhagic necrosis. Lesions in the optic
nerve and retinal degeneration occurred with organic pentavalent ar-
senical compounds. Eagle.and.Doak (1951) reported that blindness
occurred only when the nitro.or.amino groups were in the pa£a_position
of the benzene ring in relation to the arsenic radical. This effect
was not observed when the arsenic and amino groups were in the orthg
or‘mgtg'positions.

In summary, much work has been done to indicate the usefulness of
organic arsenicals to stimulate faster growth of swine and poultry.
Considerable research has been conducted to evaluate the toxicity and
effectiveness of arsenic compounds. Much work remains to be done,
however, on factors which may.alter the metabolism and the toxicity of
organic arsenicals. Two factors to be evaluated in this paper are
the reduction in water consumption and variations in quantity of die—

tary protein.

MATERIALS AND METHODS

The basic experimental plan was that each group of rats and pigs
had a companion control group which received the similar diet except
for the arsanilic acid. .The-amount of water and, in certain experi-
ments, the level of protein were varied among different groups. White
rats were used in Experiments 1 and 2. These experiments served as a
pilot study for the experiments in swine. Experiments 3, 4 and 5
utilized 4—week—old pigs to evaluate the effects of reduced water and
normal or low protein levels on arsenic toxicosis. The procedures
are summarized (TABLE 1).

In Experiment 1 the rats were fed a ration containing 24% pro-
tein. The diet for the 2nd experiment contained 8% protein and was
composed of yellow corn meal, supplemented with minerals and vitamins.
Water was reduced to 1/3 of the amount of the paired groups' water
consumption. The rats were weighed weekly. Feed and water consumption
was measured daily. All observed clinical signs were recorded, and
dead rats were necropsied.. The surviving rats were killed and necrOp-
sied at the end of the 35-day feeding period. Gross and histologic
examinations were conducted and the results recorded. The tissues taken
at necropsy included brain, stomach, small intestine, spleen, liver,
kidney, lung, and cardiac muscle. The tissues were fixed in 10%
buffered formalin solution, sectioned at 6 microns and stained with
hematoxylin and eosin, as.described in the Armed Forces Manual of
.flistologic and Special Staining Technics (1960).

10

11

TABLE 1. Summary.of experimental procedure and groups of animals
given arsanilic acid (AA)

 

 

 

Experi— Protein Days on No. of AA
ment Species ration (%) experiment Group animals (%) H20*
l Wh. rats 24 35 A 4 0.50 N
(Purina 35 a 3 ~-- N
Rat Chow) 35 B 4 0.50 1/3
35 b 3 --- 1/3
2 Wh. rats 8 35 C 4 0.50 N
(Quaker 35 c 3 --- N
Corn Meal) 35 D 4 0.50 1/3
35 d 3 --- 1/3
3 Swine 19. 10 1 2 0.10 120 ml.
(MSU Starter) 42 2 3 0.02 120 ml.
42 3 2 --- 120 ml.
4 Swine 19 42 1 3 0.02 N
(MSU Starter) 42 2 2 --- N
42 3 3 0.02 1/3
42 4 2 --- 1/3
42 5 3 0.02 1/6
42 6 2 ——— 1/6
42 7 3 0.02 l/12
42 8 2 --- 1/12
5 Swine 8 126-(42)** l 2 0.02 1/3
(Ground Corn) 126-(42)** 2 1 --- 1/3

* N = the amount of water-consumed ag_1ibitum daily. The other
groups are expressed as a fraction of N.

** The groups had been fed an 8% protein diet for 84 days prior to
addition of AA to Group 1.

12

Swine 4 to 5 weeks of age at the start of the experiment were used
in Experiments 3, 4 and 5.- Pigs in Experiments 3 and 4 were fed a
pig-starter ration containing 19% protein.

Experiment 3 was the arsenic toxicosis control group. The 2 pigs
in Group 1 were given an average of 120 ml. (60 to 240 ml.) of water
containing 64 mg. or 44 mg. (average 0.1% of ration) of arsanilic acid
orally with a dose syringe, respectively. No other water was available.
Treatment was continued until.clinical signs of blindness and ataxia
occurred. Group 2 was fed.0.02% arsanilic acid in the diet and 120 ml.
of water per pig, while Group 3 received no arsanilic acid and 120 ml.
of water per pig.

Pigs in Experiment 4, Groups 1 and 2, were allowed water free
choice, and those in Groups 3, 4, 5, 6, 7 and 8 were given amounts
reduced proportionately..

Pigs in Experiment 5-were fed an 8% protein ration composed of
ground corn, supplemented with minerals and vitamins. These pigs were
fed the 8% protein diet for 12 weeks before the 0.02% arsanilic acid
was added to the diet of Group 1.. The water allowed was 1/3 the amount
consumed by Groups 1 and 2.in Experiment 4.

Each pig was weighed, and blood was drawn from the anterior vena
cava for hematologic examination weekly. Feed consumption and water
intake were measured and-recorded on a daily basis.

At the end of the 6—week.feeding period, all animals were killed
by electrocution and necropsied. .Gross and histologic examinations
were conducted on each pig.- Liver samples were submitted to the Trace
Metals Research Laboratory, Homewood, Illinois, for analysis of arsenic

content 0

l3
Specimens collected at necropsy included brain, spinal cord, optic

nerve, sciatic nerve, eye, skin, lung, heart, stomach, small intestine,

large intestine, spleen, kidney, and liver. These tissues were placed

in l or more of the following-fixatives: Zenker's fixative, 10%

buffered formalin and Carnoy3s fluid. Sections of liver and kidney

fixed in Carnoy's fluid were.stained by the periodic acid-Schiff

method to demonstrate carbohydrates. Selected liver sections were cut

on the cryostat and stained with oil red 0 for fat identification.
Selected sections of brain and spinal cord were stained with Heiden—
hain's aniline blue stain,-thionine stain or crystal violet. The pro-

cedures used were in accordance.with the Armed Forces Manual of Histo-

logic and Special Staining Technics (1960).

RESULTS

Results of Experiments 1 and 2 are shown (TABLE 2). Reduction in
water caused loss in weight and.reduction in feed intake, which was
greater for rats in Experiment 1 (24% protein) than in Experiment 2
(8% protein). Since rats.given.an 8% protein diet consumed more feed
daily, they therefore consumed more arsanilic acid than did the rats
fed 24% protein.

Group A (24% protein diet, 0.5% arsanilic acid, and a continual
supply of water) had 3 deaths-(at 17, 18 and 25 days, respectively),*
with l rat still alive.at.the.end of the 35-day feeding period. Group
B (24% protein diet, 0.5% arsanilic acid and 2/3 reduction of available
water) had the greatest weight-losses of the groups of rats given
arsanilic acid, and all 4.rats died (at 17, 20, 25, and 25 days,
respectively). Only 1 other.rat died (in Group D on 8% protein diet,
0.5% arsanilic acid and 2/3 reduction of available water). Death
occurred on the 15th day and was believed to have been unrelated to
the arsanilic acid.

Clinical signs before death were depression, diarrhea, and pale
mucous membranes. Upon.necropsy there were hemorrhagic gastric ulcers
with free chocolatevcolored—blood in the stomach and intestinal tract.
No other gross lesions.were noticed. Histologic examination revealed

erosions and ulcers of the stomach mucosae. Hyperemia and hemorrhages

*Day of experiment.

14

15

Summary of weight changes, mortality, and feed and water
intake of rats in Experiments 1 and 2.

TABLE 2.

 

 

 

Protein Group Avg. feed Avg. H20 Avg. wt. Avg. wt. Deaths

Exp. level & No. AA intake intake (kg.2 loss and
No. (%) Animals (%) (Gm.) (ml.) 8* F** (Gm.) day***

l 24 A—4 0.5 7.7 32.8 168.2 141.8 26.2 3/4
(17,18,

25)

24 A'-4# 0.5 7.9 40.7 348.0 283.5 64. 0/4

24 a-3 --- 14.9 36.5 320.0 309.7 10. 0/3

24 B—4 0.5 5.9 9.0 171.0 125.0 46. 4/4
(17,20,
25,25)

24 b-3 -—— 9.6 14.0 355.0 289.7 65. 0/3

2 8 C—4 0.5 10.5 23.7 173.5 162.3 11. 0/4

8 c—3 --- 19.6 20.5 346.0 344.0 2. 0/3

8 D-4 0.5 7.0 9.0 174.0 146.5 27. 1/4

(14)

8 d—3 --- 14.5 10.7 300.3 287.0 13. 0/3

 

* Beginning of experiment

** End of experiment

*** Days after start of experiment
# A' was a repetition of Group A

16
were present in the brain sections examined. In Experiment 2, those
rats killed had no gross nor histologic lesions. The 1 live rat in
Experiment 1, Group A, did have ulcers and erosions in the stomach when
necropsied. The control rats (Groups A, B, C and D) lost weight with
reduction in water consumption, but no deaths occurred and the killed
animals had no gross nor histologic lesions. The controls had a large
amount of abdominal fat as compared to rats fed diets containing 24%
protein and arsanilic acid. The protocol for Group A was repeated
with larger rats, and no deaths occurred in the 35-day feeding period.
Two of the 4 rats had gross and histologic lesions of ulcers and
erosions of the stomach mucosae.

In Experiment 3, 2.pigs (Group 1) were used to produce arsenic
toxicosis. These pigs weighed 8.6 Kg. and 17.2 Kg., respectively, and
were given 44 mg. and 64 mg. of arsanilic acid daily, respectively.

This was equivalent to about 0.1% arsanilic acid in the diet. Water

was varied from 60 to 240 ml. and averaged 120 ml. daily. Both pigs
began to show posterior weakness and a staggering gait on the 8th day.

On the 10th day posterior paralysis was present and both pigs were
apparently blind. The pigs.were then given 240 ml. of water daily and
moistened feed 3 days before-they were killed and necropsied. The larger
pig drank and ate readily and regained control of his hindquarters.

The pigs still appeared to be.blind prior to necrOpsy. No gross lesions
were located. Tissue sections of the brain and spinal cord were stained
with hematoxylin and eosin.. Selected sections were stained with Mallory's
trichrome and Nissl's.stain,.as.described in the Armed Forces Manual of
Histologic and Special Staining Technics (1960). Sections of cerebrum,

especially in the temporal region dorsal to the Optic chiasma, had

l7
lesions of hyperemia, hemorrhage, neuronal degeneration with satel-
litosis and neuronophagia,.and perivascular accumulation of lymphocytes
and neuroglial cells (Figures 1 and 2). The lumbar area of the spinal
cord had hyperemia, hemorrhage,-and gliosis; also, endothelial damage
of small vessels in the dorsal horn of the gray matter was noted, with
perivascular accumulation.of.lymphocytes and neuroglial cells. The
peripheral nerve sectioned with the spinal cord had accumulations of
lymphocytes (Figures 3,.4, and 5). Retinal degeneration was not evi—
dent. The blindness was thought to be associated with the vascular
and neuronal lesion in the central nervous system. No lesions were
evident in the viscera. The arsenic content of liver samples of Group
1 averaged 5.0 p.p.m. A3203.on.a wet tissue basis (TABLE 3).

The results of Experiments 4 and 5 indicated that a reduction in
available water caused a proportional decrease in rate of gain. These
and other results are.summarized.(TABLE 3). The pigs fed arsanilic
acid gained slightly better than.their controls. The arsenic stored in
the liver generally increased-with water deprivation from 1.80 p.p.m.
in Group 1 with free choice water.(l644 ml.) to 3.30 p.p.m. in Group
5, given 240 ml. water.dai1y...Pigs in Group 7, given only 120 ml.
water daily, consumed less feed than those in Group 5, and the arsenic
stored in the liver averaged 2.70 p.p.m. Pigs in Experiment 5, given
an 8% protein ration, had.the.highest daily feed intake, but the average
storage of arsenic in the liver.was only 2.80 p.p.m. No clinical signs
of arsenic toxicosis.occurred in pigs fed the ration containing 19%
protein. The hematologic.determinations were all within the normal
range (Calhoun and.Smith,.1964).-.No gross or histologic lesions re-

lated to arsenic toxicosis were observed.

18

. ' O ' o . . . g
, , . r '_ . .
I o '
‘ . . k 0 ‘ o
o . . ‘ ' O .
. I . . ‘ . O u
p . I ‘, ‘ O .
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.' . o O
9 . ’ . t . O . .¢ 0 o . . ‘ ‘ \ ..‘
V . .
. . 9 o t. o ‘0'. ‘0. ' . .
' v . ~ .. '. Q‘. .0.
I O g 0
..n ’ I O . J
o . O " . .3 '0. O . o ' \ h ‘ .
7 0 o .J/ . . . o O ' 0 .
I . 0' C! I Y -\ . - ‘0 .
Q ‘ ' . . . ’ ' I ‘
.0 0 ' ' 0 g ; l . ‘ a
I I ' I . 'J
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. . . ' \ . o " ‘ .‘
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' ‘ 0' . . ‘ . ' o ‘
o ‘ .. ’D‘. . . .‘ '3 z . g. ‘
‘n‘no . . ,g.‘ f a _. .I’ '. 1' Q I o ’. 1 I

Figure 1. .Temporal region of cerebrum dorsal to the
optic chiasma. Satellitosis with neuronal degeneration.
Pig Experiment 3, Group 1. Hematoxylin and eosin. x 187.5.

 

Figure 2. Higher magnification of Figure 1. Notice
neuroglial cells,.lymphocytes, and neuronophagia. Hema—
toxylin and eosin. x 469.

19

 

Figure.3.. Dorsa1.horn of lumbar spinal cord. Peri-
vascular.accumulation of.1ymphocytes and neuroglial cells
(A). Satellitosis and neuronal degeneration (B). Pig Ex-
periment 3, Group 1. Hematoxylin and eosin. x 187.5.

'

 

Figure 4...Higher magnification of Figure 3, Part A.
Notice neuronal degeneration, lymphocytes, and neuroglial
cells. Hematoxylin and eosin. x 469.

20

 

cu .. ‘
.II
I

Figure 5. .Higher-magnification of Figure 3, Part
B. Dorsal horn-of-lumbar.spinal.cord. Perivascular
accumulation of lymphocytes and neuroglial cells. Hema-
toxylin and eosin. x 469.

21

TABLE 3. Summary of weight gains, liver arsenic trioxide content,

and water and feed intake in pigs

 

 

 

 

Group Avg. feed. Avg. H 0 Avg. wt. Avg. wt. Liver arsenic
Exp. & No. AA intake inta e (Kg.) gain oxide content
No. Animals (%) (Kg.) (ml.) 9* F** ((Kg.) (p.p.m.)
4 1—3 0.02 1.10 1644 3. 18.5 15.1 1.80
(19%
prot.) 2-2 --- 1.10 1833 2. 14.5 11.9 0.40
3-3 0.02 1.00 555 4. 13.8 9.8 1.90
4-2 -—— 1.06 591 3. 12.5 8.9 0.60
5-3 0.02 1.45 240 12. 25.1 13.1 3.30
6-2 --- 1.11 240 11. 24.3 13.0 0.10
7-3 0.02 1.04 120 11. 22.5 11.2 2.70
8-2 -—— 0.86 120 12. 20.4 8.2 0.20
5 l—2*** 0.02 2.04 480 18. 23.6 5.0 2.80
(8%
prot.) 2-1*** ——— 1.81 480 16. 21.3 5.0 0.45
* Beginning of experiment
** End of experiment
*** Groups had been fed 8% protein diet for 84 days prior to

addition of AA to Group 1.

ar—
I

 

22
Pig 2 in Experiment 5, Group 1, fed the diet containing 8% protein,
had clinical signs resembling arsenic toxicosis, which included ataxia
and anorexia; but the pig continued to drink water when it was supplied.

At necropsy no gross nor histologic lesions were evident.

DISCUSSION

Arsanilic acid toxicosis was produced in rats in the first experi—
ment. A sublethal level of arsanilic acid (0.5%) and a 24% protein
diet were common.factors...The-effect of reduction of water consump-
tion was not consistent. .Three of 4 young rats died when fed a 24%
protein diet containing 0.5% arsanilic acid and water §g_libitum (Ex-
periment 1, Group A), while older rats given the same treatment sur-
vived (Experiment 1, Group A'). These older rats, however, did have
lesions similar to those seen in the younger rats that died. Longer
administration of arsanilic.acid might have caused death. Rats given
diets containing 8% protein consumed more feed and, consequently, more
arsanilic acid, but toxicosis did not occur. Lower levels of protein
may therefore be a factor in prevention of arsanilic acid toxicosis of
rats. Rats fed a diet containing 24% protein drank more water than
those given an 8% protein diet. This was to be expected, since 1
calorie of protein requires 3 m1. of water for the elimination of urea
and sulfates formed from protein metabolism (Rupel, 1929).

Pigs fed a 19% protein ration containing 0.02% arsanilic acid
developed no signs of toxicosis, even though water consumption was re-
duced to as little as 1/12 normal for 6 weeks. Arsenic levels in the
liver were higher in pigs in which water consumption was restricted.
Pigs given 1/6 of the controls'.daily water consumption had the highest
levels of arsenic in.the.liver.. Food consumption was affected when
water consumption was further reduced, and this was reflected in the

23

24
smaller increase in hepatic-arsenic content.

The growth-stimulating-effect of arsanilic acid was indicated by
the greater rate of gain of pigs given arsanilic acid as compared to
the controls. Although restriction of water generally decreased food
intake and rate of gain, decreases were not as severe as expected.

Oliver and Roe (1957) reported that 8 p.p.m. A5203 was present
in the livers of swine with arsenic toxicosis. The pigs with signs

of arsenic toxicosis in Experiment 3 had an average of 5 p.p.m. A3203

 

 

in liver tissue. The small difference in liver arsenic content between
those pigs with signs and.lesions of toxicosis (5 p.p.m. A3203) and

the pigs receiving arsanilic acid in the feed and limited water

(3.3 p.p.m. A3203) would tend.to indicate that, under certain field
conditions, arsenic toxicosis.might occur. This work further estab-
lishes the safety of arsanilic.acid for swine when properly used but
indicates that accumulations of arsenic will occur in the body when

water intake is reduced.

SUMMARY

Reduction in water consumption and variation in the quantity of
dietary protein were.evaluated as factors affecting the toxicity of
arsanilic acid for rats and swine.

In rats, arsanilic acid.toxicosis was produced with a diet contain-
ing 24% protein and 0.5% arsanilic-acid. A 2/3 reduction of water de-
creased the length of time.for toxicosis to appear. Lesions included
gastric ulcers and.cerebral.hyperemia and hemorrhages. Rats fed an 8%
protein diet and 0.5% arsanilic acid and control rats had no apparent
lesions.

Pigs given 44 mg. or 64 mg. of arsanilic acid orally daily for 8
days by dose syringe and with limited water available developed signs
and lesions of arsenic toxicosis. The clinical signs were posterior
paralysis and4blindness. .The cerebrum, especially in the temporal
region dorsal to the—optic.chiasma, had lesions of hyperemia, hemor-
rhages, neuronal degeneration with Satellitosis and neuronophagia, and
perivascular accumulation.of lymphocytes and neuroglial cells. The
lumbar area of the spina1.cord had hyperemia, hemorrhage and gliosis.
Also noticed was endothelia1.damage of small vessels in the dorsal horn
of the gray matter, with.perivascu1ar accumulation of lymphocytes and
neuroglial cells. The peripheral nerve sectioned with the spinal cord
had accumulations of.1ymphocytes.

The pigs fed.rations.containing 0.02% arsanilic acid with levels
of 8% and 19% protein and given water varying quantitatively from

25

-26
120 m1. daily to free choice (average, 1644 ml. daily) had no signs or
lesions of arsenic toxicosis.

Analysis for arsenic trioxide (A8203) stored in the liver revealed
averages of 5 p.p.m. in pigs with clinical signs and lesions of arsenic
toxicosis, 3.3 p.p.m. in pigs.with 0.02% arsanilic acid in the diet and
240 ml. water daily, and 1.80.p.p.m. in the control pigs fed 0.02%
arsanilic acid and water-§g;libitum. The control pigs averaged less
than 0.60 p.p.m. A3203.in.the liver.

It was concluded that.both-the quantity of water and the level of
protein may be factors in arsenic toxicity. The results also confirm

previous reports of the safety of arsanilic acid when used as recommended.

REFERENCES

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Special Staining Technics. Washington, D.C.

Calhoun, M. L. and Smith,.E. M.. 1964. Diseases of Swine: Hematology
and Hematopoietic. 2nd ed., edited by Howard W. Dunne. 'Iowa
State Univ. Press,.Ames, Iowa.

Carpenter, L. E. 1951. Effect of 3—nitro-4-hydroxy-phenyl arsonic
acid on the growth of swine. Archives of Biochem. and Biophys.,
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Carpenter, L. E. and Larson, N. L. 1952. Swine dysentery treatment
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Coulson, E. J., Remington, R. E. and Lynch, K. M. 1939. Metabolism in
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Davis, J. W. 1961. Studies on swine dysentery. J.A.V.M.A., 138:
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Eagle, H. and Doak, G. 0. 1951. The biological activity of arseno
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from Western Feed and.Seed.

Frost, D. V. 1952. "Safety first" in use of arsonic compounds in feed.
Feed Age, 2: 30—31.

Frost, D. V., Perdue,.H. S. and Overby, L. R. 1961. Arsenicals in
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Frost, D. V. and Spruth, H. C. 1956._ Arsenicals in feeds. Symp. of
Medicated Feeds, Medical Encyclopedia, New York, N.Y., 136—149.

27

 

 

.28

Garner, R. J. 1961. .Veterinary Toxicology, 2nd ed. Williams & Wilkins
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29

Overby, L. R. and Frost, D. V. 1960. Excretion studies in swine fed
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VITA

The author was born June 26, 1937, in Fairfield, Iowa, and
attended public school in the same city. In the fall of 1955 the
author enrolled at Parsons College and attended for one year. Pre-
veterinary work commenced in the fall of 1956 at Iowa State College.
In 1958 the author was accepted in the Veterinary College at Iowa
State University and graduated in May, 1962.

During the summer the author worked for a small animal practi-
tioner in Davenport, Iowa. That fall he accepted a position with
Dr. Harold Weimner at Riverside, Iowa in what was predominantly a
swine practice. In 1964 the author accepted a position with the
Ambulatory Clinic at Michigan State University. At this time he
also began graduate training in pathology at the university. In the
spring of 1966 the author accepted a position in the Department of
Anatomy, teaching a course in large animal anatomy and applied and

surgical anatomy.

30

 

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