EPA
560/11-80-
022
FAILURE TO PRODUCE ARSENIC NEUROTOXICITY
IN THE RAT
AN EXPERIMENTAL STUDY
MAY 1980
FINAL REPORT
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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EPA 560/11-80-022
May 1980
FAILURE TO PRODUCE ARSENIC NEUROTOXICITY
IN THE RAT
AN EXPERIMENTAL STUDY
Herbert H. Schaumburg
Institute of Neurotoxicology
Albert Einstein College of Medicine
1. Historical Backgroud
2. Experimental Neuropathology
3. Human Neuropathology
U. Purpose and Justification of this Study
5. Methods
6. Rusults
7. Summary and Conclusions
8. Appendix - Figure and Table
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DISCLAIMER
This project has been funded with Federal funds from the
Environmental Protection Agecny under contract number
68t-01-3859 . The content of this publication does not
necessarily reflect the views or policies of the U.S.
Environmental Protection Agency, nor does memtion of trade
names, commercial products, or organizations imply endorse-
ment by the U.S. Government.
Yii
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abstract
Prolonged, low-level exposure to inorganic arsenic can produce
peripheral neuropathy in humans, and is a serious industrial
health hazard. The clinical expression of arsenic neuropathy
is similar to other toxic neuropathies of the dying-back type.
No satisfactory animal model of arsenic neuropathy has been
devised.
Rats underwent weekly intraperitoneal injections with solutions
of arsenic trioxide. Strengths of 2mg/kg, 5mg/kg, lOmg/kg and
15mg/kg were used. The 15mg/kg animals died shortly after
receiving the injection. The other animals survived and, after
eighteen months, appeared normal. Histopathological study of
the peripheral and central nervous systems of these animals was
unr emarkab1e.
It appears that the rat is not the appropriate species for the
study of inorganic arsenic neurotoxicity.
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2
I. HISTORICAL BACKGROUND
Inorganic arsenic was utilized as a form of therapy by the ancient Greek
and Roman physicians. Epidemics of polyneuritis in the past suggest the possibility
of widespread arsenical poisoning. An epidemic of 40,000 cases of arsenisrti occurred
in France in 1828 when arsenious acid (arsenic trioxide dissolved in dilute hydrochloric _
acid) was accidentally introduced into wine and bread. In England in 1900, there
was an epidemic of arsenic neuropathy in which an estimated 6000 cases and
approximately 70 deaths occurred. This outbreak or arsenism resulted from the
use of arsenic-contaminated sugar in the preparation of beer. A comparable
outbreak followed the use of malt that was contaminated with arsenic as the
consequence of having been dried with coke gas. Arsenic trioxide is a product
of smelting operations, and it is the starting material for synthesizing most arsenical
compounds. In the natural environment, most arsenicals degrade or weather
to form arsenate.
Tolerance to arsenic varies, and this has been attributed to individual idiosyn-
crasies. Levels of 0.05 to 0.30 ppm are normally found in most tissues, similar
to levels found in the rabbit. Arsenic eating habitually took place in Styria and
Tyrol. Persons who ate arsenic could tolerate dosages of approximately 5 mg
per day; more habitual arsenic eaters could tolerate 500 mg in a single oral dose.
The medicinal use of Fowler's solution was more common in the past, and excessive
administration of such arsenical medications led to a neuropathy. In addition
to accidental poisoning as a result of contamination of bread, wine, or beer, arsenical
intoxication has been due to occupational exposure in a number of industries
for example, power production, leather manufacturing, paint and dye production,
and the preparation of insecticides.
Biological and Clinical:
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Arsenic neuropathy is the most common type of metal neuropathy and,
by its clinical expression, is clearly of the dying-back type. Many times, the
source of the arsenic remains a mystery. With a mild neuropathy, the patient
may mention only the presence of mild paresthesias, usually of the feet. With
severe involvement of the peripheral nerves, there is a severe sensory of sensorimotor
polyneuropathy with a stocking-glove pattern of hyperesthesia or anesthesia
and distal weakness, both of which may preclude ambulation. Often the patient
will complain of intense pain or painful paresthesiase, tenderness, and swelling
of the hands and feet, more troublesome in the lower limbs than in the upper.
Mild neuropathies may be painless. When the upper limbs are involved, the medial
and ulnar nerves are more affected than the other upper limb nerves. Paralysis
of the facial nerve and motor portion of the trigeminal nerve has also been reported.
At times, with chronic poisoning, there is a brownish discoloration of the skin
that, on the trunk, may assume a teardrop appearance with a dermatome distribution.
The skin lesions have been described a herpetiforn or pemphigoid. The soles,
and at times the hands, become darkened and hyperkeratotic, and these changes
can be followed by skin cancer. Another variation is that the hands or feet become
swollen, blotchy, reddened or livid, and hyperhidrotic. The nails are sometimes
brittle and thin, with the appearance of transverse whitish striae (Mees1 lines).
Mees' lines appear approximately a month after the ingestion of arsenic.
Chronic arsenical intoxication, either accidental or intentional, is insidious
in onset and is characterized by nonspecific symptoms such as malaise, nausea,
anorexia, generalized weakness, intermittent diarrhea, and constipation. Upper
respiratory symptoms also can be present, as well as increased salivation and
stomatitis. Keratosis of the palms and soles with increased pigmentation (arsenical
melanosis) is characteristic of chronic exposure to arsenic. Heptomegaly, jaundice,
and changes in the liver compatible with cirrhosis can be found. The toxic effect
on the bone marrow leads to anemia, usually of the aplastic type.
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k
2. EXPERIMENTAL NEUROPATHOLOGY:
The structural changes of the peripheral nervous system caused by experimental
arsenic intoxication have not been studied very thoroughly. In a study of spinal
cords of dogs after arsenic intoxication performed in 1883', vacuoles and other
abnormalities appeared in anterior horn cell neurons. These probably represent
2
fixation artifact. Except for one study in pigs , in which no histology was illustrated,
no satisfactory animal model has been devised.
3- HUMAN NEUROPATHOLOGY:
Erlicki and Rybalkin's study of two human cases in 1892 probably represents
the most extensive and reliable evaluation of the morphologic changes in arsenic
3
neuropathy . The cases were carefully studied clinically over many months,
and extensive pathologic studies were done in one case. In both cases, the time
of the accidental ingestion of arsenic was known. In addition to the initial abdominal
pain, vomiting, and subsequent delirium, pain described as burning, searing, tearing,
stabbing, and cutting and of extreme severity developed in about one week. The
pain was associated with decreased touch-pressure, temperature discrimination,
and pain sensations, and was located in the soles of the feet, toes, lowerlimbs,
hands, and fingers. It was much accentuated by putting weight on the feet or
scratching the sole. Muscle weakness began 3 to k days after arsenic ingestion •
and reached its greatest severity at approximately 12 days. The greatest weakness
was in the distal muscles of the lower limb, more in small foot muscles and peroneal
muscles than in anterior tibial, calf, or thigh muscles. In the more severe case,
more proximal muscles were affected also. In one case, recovery of muscle strength
began at six weeks in proximal muscles. In the milder case, by one and a half
years muscle strength had returned except for the extensor muscles of the ulnar
three fingers; in lower limbs also, improvement had occurred so that walking
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was possible with a cane. The more severely affected patient died from pneumonia
during the prolonged convalescence. Although incontinence occurred during the
period of delirium, bowel and bladder function were not affected. Tendon reflexes,
particularly of the lower limbs, were absent. In the milder case the knee reflex
returned a year and a half after the exposure.
At autopsy in the more severe case, pathologic changes were found in the
spinal cord and peripheral nerves. Anterior horn cells were decreased in number
in particular groups of the ventral gray matter and also diffusely. None of the
remaining cells was normal: they had lost their angular contours, were rounded,
and often lacked processes; the cytoplasm was altered; and the normal "stripes"
had degenerated into granular yellow or yellow-brown pigment. This last change
may reflect nothing more than the presence of lipofuscin, an abnormality occurring
in spinal cords unaffected by specific diseases. Nuclei were ragged and sometimes
pale. In the cervical and lumbar enlargements of the spinal cord there was no
marked abnormality except that posterior and anterior columns were markedly
thinned and had decreased numbers of myelinated fibers. Only the radial and
peroneal nerves were evaluated. The majority of myelinated fibers were abnormal,
but the morphologic changes were not described in detail. In the authors' opinion,
some fibers had remained without degeneraton, some were degenerating, and
others were regenerating. Unequivocal evidence of both central and peripheral
pathologic changes was thought to be present.
Longo and co-workers reported extensive degeneration in distal ramifications
of peripheral nerve fibers, as seen in wallerian degeneration. In a series of 708
patients with neuropathy, Chhuttani and co-workers (1967)"* found 40 in whom
the neuropathy could be blamed on arsenic. Biopsy of a digital nerve of the toe
showed increased cellularity, increased thickness of perineurium, and a decreased
number of and degeneration of myelinated fibers.
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4. PURPOSE OF THIS STUDY AND JUSTIFICATION:
This study was designed to produce chronic inorganic arsenic intoxication
in rats, resulting in a peripheral neuropathy resembling the human neuropathy
associated with prolonged low level environmental or industrial exposure. Our
purpose was to study the spatial temporal evolution of peripheral and central
nervous system disease. No satisfactory animal model of human arsenic intoxication
exists (see background).
Several benefits would stem from a morphological study of an experimental
animal model:
1) The distribution of the neuropathological changes would indicate
the vulnerable areas of the nervous system, and greatly facilitate clinical evaluation
of humans exposed to arsenic. This would be especially useful in suggesting the
proper clinical test used in a screening operation amongst workers exposed to
arsenic.
2) The character of the neuropathological changes might suggest the
nature of the biochemical abnormality underlying arsenic neurotoxicity, and facilitate
prevention or treatment of arsenic neuropathy.
5. METHODS:
Eighteen young male adult Sprague-Dawley rats were used in this study.
Fifteen animals underwent weekly intraperitoneal injections with solutions of
arsenic trioxide. These 15 animals were divided into 4 groups depending on the
strength of the solution injected 15 mg/kg, 10 mg/kg, 5 mg/kg, and 2 mg/kg.
Three animals were used as controls, and received weekly injections of normal
saline. All animals were allowed food and water ad libitum, examined daily and
weighed weekly.
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Perfusion and Tissue Preparation:
At the completion of the period of intoxication, each animal was anesthetized
with sodium barbitone containing heparin, the chest opened and the animal perfused
with fixative (phosphate-buffered 4% paraformaladehyde for 1 minute at lOOmm/Hg
pressure followed by phosphate-buffered 5% glutaraldehyde at 100-180 mm/Hg
for 15 minutes) by a cannula, placed into the apex of the heart, opening into the
aortic arch, and draining via the opened right atrium. Tissue was sampled (vide
infra) and immersed in Dalton's chrome osmium tetroxide for one hour of postfixation.
After alcohol dehydration, epon infiltration, block hardening and trimming, one
micrometer sections were cut with a glass knife in a Porter Blum MT2B, mounted
on glass slides and stained with a 1% aqueous solution of toluidine blue with 0.5%
borax for I minute at 300°C. These slides were examined with a light microscope.
To prepare teased PNS fibers, dehydrated nerves were infiltrated with epoxy
resin, and the epineurium and perineurium removed. Single fibers were dissected
apart with needles, the fibers mounted on glass slides, hardened and a cover slip
mounted with epoxy resin. The preparation was then be examined by light microscopy.
Standard Tissue Areas Sampled: These include the plantar, tibial and sciatic
nerves, dorsal and ventral lumbar roots, dorsal root ganglia, multiple levels of
the spinal cord, medulla oblongata, cerebellum, pons, hypothalamus, optic nerve,
lateral geniculate body and superior colliculus. We have observed axon changes
in other toxic neuropathies at all of these sites.
Tissue arsenic levels - Samples of urine and hair from intoxicated animals were
submitted on two occasions to the Poisonlab (1469 S Holley St., Denver, Colorado)
for arsenic analysis.
6. RESULTS: (See appendix)
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1) 2 mq/kq - 4 rats intoxicated at this level for 18 months displayed normal
growth, and behavior. Microscopic examination after sacrifice revealed no abnormalities
in the nervous system. Urine arsenic levels (Table I of Appendix) after 9 months
were 2100 meg/liter (2920 meg arsenic per gram of creatinine).
2) 5 mq/kq - 4 rats intoxicated at this level for 18 months displayed normal
growth and behavior. Microscopic examination after sacrifice revealed no abnormalities
in the nervous system. Urine arsenic levels (Table I of Appendix) after 9 months
were 2500 meg/liter (5680 meg arsenic per gram of creatinine).
3) 10 mq/kq - 4 rats were intoxicated at this level and died after 4 months.
Autopsy revealed only bronchial pneumonia and hepatic congestion. A fifth rat
was administered 10 mmg/kg and survived for 12 months, with normal growth
and no clinical findings. Microscopic examination (illustrated in Fig. I of Appendix)
of the nervous system revealed no significant changes. Urine arsenic levels (Table
I of Appendix) of this animal after four months revealed 2800mcg/L (9330 meg
of arsenic per gram of arsenic).
15 mq/kq - Two rats were intoxicated at this level and died suddenly
after the second injection. Autopsy revealed hemorrhages in the upper gastrointestinal
tract and kidneys.
5) Controls - Three control rats received weekly saline injections for 18
months and at time of sacrifice displayed no lesions in the nervous system.
7. SUMMARY AND CONCLUSION:
Intoxication of rats with inorganic arsenic for 18 months has failed to produce
nervous system damage despite high levels of total body arsenic. It appears that
prolonged intoxication of rats with levels exceeding 5 mg/kg per week is extremely
difficult because of high mortality. We succeeded in maintaining one animal
for a year at the 10 mg/kg and, despite formidible levels of total body arsenic,
no neurotoxicity developed.
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There are two possible explanations for the failure of this study to produce
neurotoxicity. One is that the rat is immune to the neurotoxic effects of arsenic
because of metabolic factors, eg. enzymes necessary for neural maintainence
in the rat nervous system are not affected by arsenic. A second explanation
is that the life span of the laboratory rat (30 months) is two short to allow adequate
prolonged, low-level accumulation of arsenic trioxide sufficient to be neurotoxic.
I favor the later explanation, and suggest that future studies of low-level inorganic
arsenic intoxication be done in young kittens, and pursued over a 4-5 year period.
References
1. Popw, N.: Virchows Archir (Path. Anat. Physiol.), 93, 351, 1883.
2. Harding, Lewis, G. and Done, J.T.: Vet. Rec., 83, 560, 1968.
3. Erlicki, A. and Rybalkin: Arch. Psychiat. Nerven Kranke., 23, 861, 1892.
k. Longo, P.W., Lemmi, 0., Giorgi, D. and Nasser, J.: Rev. Neuropsiqiatr.,
28, 52, I 965.
5. Chhuttani, P.M., Chawla, L.S. and Sharma, T.D.: Neurology, 17, 269, 1967.
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Appendix
Figure I: Sections of the tibial nerve near caif muscles (a,b) and the gracile
fasiculus (c) from rat intoxicated for 12 months with 10 mg/kg/week. These
sections display no abnormal features.
Table I: Report from Poisionlab on urine and hair arsenic analysis of h rats from
this study. Note highest levels in animal 1(10 mg) and lesser levels in animals
2 (5 mg), 3 (2 mg) and k (control).
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01SONLAB //
6227 lomg
6228 5mg
6229 2rag
6230 Control
6231 lomg
6232 5mg
6233 2mg
6234 Control
SPECIMEN #
//I Urine
//2 Urine
// 3 Urine
// 4 Urine
//I Hair
#2 Hair
//3 Hair
#4 Hair
ARSENIC
2800 mcg/L
2500 mcg/L
2100 mcg/L
Less than 20 mcg/L
29 mcg/gm (ppm)
20 meg/gm (ppm)
12 mcg/gm (ppm)
Less than 1 mcg/gm
TABLE I
CREATININE
ARSENIC/gm CREATININE
.3 gms/L
9330 meg Arsenic
gm Creat inine
.4 4 gms/L
5680 meg Arsenic
gm Creatinine
.7 2 gms/L
2920 mcg-Arsenic
gm Creatinine
.4 gms/L
Less than 50 meg
gm Creatinine
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APPENDIX
CI
FIG I
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completinz)
1. REPORT NO.
2.
3. * raco-^uy^up
4. TITLE AND SUBTITLE
Failure to produce arsenic neurptoScicity
in the rat.An Experimental -Study.
5. REkohT DATE
May 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR
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