Neural lesions induced in ducklings by isonicotinic acid hydrazide and semicarbazide hydrochloride

EXPERIMENTAL

AND MOLECULAR

Neural

Lesions Hydrazide

WILLIAM Department

W. of

PATHOLOGY 4,438-448

Induced and

CARLTON, Nutrition

(1965)

in Ducklings

by

Semicarbazide CHARLES

E. HUNT,

.4ugust

Acid

Hydrochloride’ AND

and Food Sciences, Massachusetts Cambridge, Massachusetts Received

lsonicotinic

PAUL M. Institute

NEWBERNE of

Technology,

17, 1964

A number of speciesof the genusLathyms produce seedswhich are poisonouswhen consumedby animals or man (Selye, 1957). Signs that develop in poisoned animals are varied; they include neurological alterations (Gardner and Sakiewicz, 1963), skeletal deformities (Geiger et al., 1933), and dissecting aneurysms of the aorta (Ponseti and Baird, 1952). Stockman (1917) reported that Bonnet monkeys (Macacus sinicus) force-fed extracts of Lathyrus spp. developed paralysis which was accompanied by morphologic lesions in the brain and spina cord. Ponseti and Baird (1952) observed paraplegia in rats with severe kyphoscoliosis and cord compression.Some investigators consider the spastic paraplegia observed in experimental animals secondary to the bone lesions, while others believe it a primary neurologic lesion (Diaz et al., 1957). The active principal of the sweet pea (Lathyrus odoratus) was identified in 1954 by Schilling and Strong (1954) as beta-amino-propionitrile (BAPN). When fed to animals, this substanceproduces skeletal deformities (Bachhuber et al., 1955), aortic aneurysm with rupture (Wawzonek et al., 1955; Lalich et al., 1957), and neurologic alterations (Lalich et al., 1956; Barnett et al., 1957). Compounds similar chemically to BAPN have been studied for their lathyrogenic activity. Bachhuber et al. (1955) reported that the administration of 0.3% of o-p’, iminodipropionitrile (IDPN) to rats resulted in nervous signs accompaniedby histologic lesionsin the anterior horn cells of the spinal cord and in the Purkinje cells of the cerebellum. These lesions as well as paresis were confirmed by Hartmann et al. (1958). Sanchez-Martin (1959) reported that chronic administration of IDPN produced paralysis of the hind limbs with necrosisof spinal motor neurons in 100% of Sprague-Dawley rats. Certain non-nitriles have been shown to produce lesions which are similar to those of lathyrism. Weanling rats fed semicarbazidehydrochloride (SCH) developed gross skeletal lesions similar to those seen after ingestion of BAPN or sweet pea meal (Dasler, 1958). Milliser and Dasler (1959) described skeletal and aortic lesions in weanling rats fed semicarbazide hydrochloride and acetone semicarbazone. Cameron (1961) reported lathyrism-like changesin chicks fed SCH or acetone semicarbazone 1 Contribution 623 from the Department of Nutrition and Food of Technology, Cambridge, Massachusetts 02139. Supported in part by grant HE-08460-01 from the National Maryland. 438

Science, Institutes

Massachusetts of

Health,

Institute Bethesda,

NE~ROLATHYRISM

IN

DUCKLINGS

439

at a dietary in Ayl&urg

level of 0.1%. More recently, Cameron ( 1962) described a kg Paresis ducklings fed 0.1% SCH. He observed, at the end of three weeks, degeneration of the anterior motor horn cells and necrosis of spinal motor neurons. During investigations of the responseof Pekin ducklings to non-nitrile lathyrogenic substances,lesionswere observed in the central nervous system which appear to differ from those reported previously (Cameron, 1962). Results of feeding non-r&rile compounds isonicotinic acid hydrazide INAH and SCH and substancesthought to have protective activity against neural damage are reported here. MATERTALS

AND METHODS

Male Pekin ducklings were allotted at random and placed on the various experimental diets within 24 hours after hatching. They were housed in an electrically heated battery brooder and were given feed and water ad Zibiturn. They were observed daily and weighed at weekly intervals. In most trials 10 ducks were allotted to each test diet and 5 control birds received chick-starter mash. Most trials were continued for 3 weeks. Chemicals tested were incorporated into a starter mash (Worthmore Feed Company, Waltham, Massachusetts) and stored in metal containers. In Trial 1 ducks were fed the starter mash containing SCH” and INAH” at two levels. The protective activity of carbonyl-donating substances,salicylaidehyde: and nt-glyceraldehyde4 were studied in Trial 2. These substanceswere added to test diets which contained SCH. m-thyroxine was added to diets which contained SCH or TNAH in Trial 3 and thiouracil was similarly studied in Trial 4 (Table II). Birds that became seriously disabled or moribund were killed by decapitation and tissueswere fixed in neutral-buffered formalin. Ducks killed at the end of the experimental periocl were handled in a similar manner. Selected tissues were prepared for paraffin sections, cut at 5 to 7 u on a rotary microtome and stained by hematoxylin and eosin for histopathological examinations. RESULTS Trial 1 Semi~rbazide and isonicotinic acid hydrazide, as 0.170 of the diet, were found to be extremely toxic to young ducklings. Growth was depressed,although early in the experiment the ducks consumed adequate amounts of the diet, Feed consumption decreasedas nervous signs developed. Using weight gain as the criterion, SCH appeared to be slightly more toxic than INAH. At the end of the third week mortality was 50% in each of the 2 groups. When the diet contained 0.0.5% SCH or INAH, growth was markedly reduced, although clinical signs of neural involvement were minimal. SCH, O.OSF. appeared to be more toxic than INAH. Eight of 10 birds fed this level of SCH died during the 4th week (Table I). Ducks fed lathyrogenic diets (0.12 ) presented nervous signs after 10-12 days of feeding. The birds were ataxic and exhibited tremors during voluntary motor movements, especially noticeable when ducklings attempted to eat and drink. The signs were exaggerated when the ducks were disturbed. Neur&gic disturbances were pro” Fisher Scientific Company, Fair Lawn, P\‘ew Jersey. 3 Eastman Organic Chemicals, Rochester 3, New York. 4 Nutritional Biochemicals Corporation, Cleveland, Ohio.

a b 0 d

0.5$~

SA”

GAc

0.5%

0.5%

Eight birds died before SA = salicylaldehyde. GA = oz-glyceraldehyde. Second week average.

GA,

Trial 2 Mash control SCH, 0.1% SCH, 0.1% + SA, 0.5% SCH, o.l’$ +

Trial 1 Mash control SCH, 0.1% SCH, 0.05% INAH, 0.1% INAH, 0.05%

Treatment

4th week

44.6 54.5 39.6

10

51.5 56.3 53.1

10 10 11

weights.

57.0 60.6 54.4

s 10 10

10 10

10

57.2 56.2

^..~ Starting km)

Average

OF FEEDING

5

Number of birds

RESULTS

SCH INAH

666.6 90.0 257,la

855.0 114.3 118.5

255.7cc 131.2 277.6

803.8 93.2

Final (m)

weight

AND

I

+

0 90.0 0

0 70.0 50.0

:

10.0

changes

+ -

+ + -

+ -

-

+ + -

+

+ + +

-

-

+ -

:

-

Spinal cord

+ + -

Lesions

-

+

+

+ +

Optic lobes

SALICYLALDEHYDE

Cerebellum

NucIear

WITH

-

+

0 50.0 0 50.0

-

Spinal cord

SUPPLEMENTATION

Mortality (9%)

AND

TABLE

-

-

-

A-

+

--

Cerebellum

Demyehnation

-

-

--

+

+

-

Optic lobes

% 0

NEUROLATHYRISM

IN

DUCKLINGS

441

gressive and the ducks were unable to maintain equilibrium. They would fall and lie on their sides and make paddling movements with their limbs. After the ducklings were down they could not return to a standing position, nor could they maintain a standing position when placed upright. They were removed from the experiment soon after these signs were observed. No deaths attributable to vascular rupture were observed in any of the experimental group. Significant gross lesions were not observed in the CNS of ducks fed SCH or INAH. Microscopic lesions were present in the cerebellum, lumbar spinal cord, optic lobes and the medulla oblongata. In the cerebellum of SCH-fed ducklings striking nuclear alterations were observed in the Purkinje motor cells. Nuclear chromatin was present in the form of granules aggregated in the center of the nucleus in an inclusion-body fashion with a clear halo between the chromatin mass and the nuclear membrane. Nucleoli were not discernable microscopically. Cells were also observed in which nuclear lysis had occurred. The percentage of Purkinje cells exhibiting nuclear changes varied among the birds with as many as 90cjc1of the cells affected in some ducklings. Demyelination was minimal to absent. The cerebellum and spinal cord of ducks fed SCH or INAH contained many degenerating motor neurons with basophilic cytoplasm and eccentrically placed, pyknotic nuclei. A few neurons with similar morphology were observed in sections of the cerebellum from control ducks. Neurons with similar nuclear changes were seen in cell groups in the medullary portion of the cerebellum, in the medulla oblongata, the optic lobes, and in the motor cells of the ventral horn of the spinal cord (Fig. 1). Striking microscopic lesions were observed in both the white and gray matter of the CNS of ducks fed INAH. Nuclear changes similar to those described above for SCH-fed ducklings were present, but an even more pronounced lesion was a noninflammatory demyelination (Fig. 2). In descendingorder of severity, demyelination was a prominent lesion in the following areas of the CNS: cerebellum, dorsal funiculi of spinal cord, medulla oblongata and optic lobes. Although demyelination was most severe in the medullary portion of the cerebellum (Fig. 2), in many of the birds it extended to the tips of the folia. In some ducks the folia involvement was of such severity that the white matter collapsed upon sectioning so that the granular layers of the cerebellar cortex were separated by only a very narrow strip of white matter. Glial proliferation and neuronophagia were not observed. Trial 2 Growth was essentially the same for the groups which received SCH plus salicylaldehyde as for the group which received SCH alone (Table I). There were fewer deaths in the former group. Salicylaldehyde alone depressedgrowth but no CNS lesions were detected microscopically. nL-Glyceraldehyde, as O.O5c/(,of the diet, reduced by one-half the growth of the test animals at the end of the 2nd week. Its addition to the SCH diet reduced growth further and mortality was higher than that observed in the SCH diet (Table I). Microscopic examination of sections of brain and spinal cord of ducks fed SCH + nr-glyceraldehyde or SCH + salicylaldehyde revealed lesions similar to those present in the ducklings which were fed SCH alone. Trial 3 nn-Thyroxine, 100 mg/kg of diet, proved to be extremely toxic to young ducks: none survived the 2nd week. There were no alterations in the tissue responseto SCH

442

W.

W.

CARLTON,

C. E.

HUNT,

AND

P. M.

NEWBERNE

INAH due to the addition of m-thyroxine to the respective diets. The combinatic ons INAH + thyroxine and SCH + thyroxine were less toxic than thyroxine ale3ne ‘able II). Because of the results observed with the first level of feeding, 2 additio nal )ups were fed thyroxine-supplemented diets. A group of 10 ducks fed mash wh ich

FIG.

horn

1.

exhibit

Lumbar typical

spinal cord of duckling fed 0.1% SCH for 3 weeks. chromatin aggregation and halo formation. Hematoxylin

Motor neurons of ventra1 and eosin stain X 1000.

NEUROLATHYRISM

IN

DUCKLINGS

443

con .tained 50 mg/kg of diet weighed 218 gm at the 2nd week; there was 50% r nortali ty. There were no deaths in a thyroxine group which received 25 mg/kg of (liet, but the average weight was only 230 gm after 2 weeks on the diet compared to 366) gm for controls.

F KG. 2. Cerebellar ax01 n loss is present stail 1 x 400.

medulla of duckling fed 0.1% INAH for 3 weeks. Severe demyelination and and extends into the folia between the granular layers. Hematoxylin and f

01 Second week. b TH = m-thyroxine. C Two birds killed d Third week. e TU = thiouracil. f Four birds killed

tissue.

tissues.

for

for

Trial 4 Mash control SCH, 0.1% SCH, 0.1% + 0.275~ TU’ INAH, 0.1% INAH, 0.1% + 0.27& TU TU, 0.2%

Trial 3 Mash control SCH, 0.1% SCH, 0.1% + 100 mg TH,‘kg INAH, 0.1% INAH, 0.1% + 100 mg TH/kgh TH, 100 mg/kg

Treatment

RESULTS

OF

10 10 10 10

10

5

9 9 9 9 10 10

Number of birds

Average

54.0 60.6 SO.6 54.5 51.7 $9.2

56.3 53.6 558 52.8 49.3

51.0

Corn)

Starting

SUPPLEMENTATION

OF

TABLE DIETS

0 70.0 60.0

50.0 100.0 0

f

131.2 404.3

0 55.6 100.0 11.1 77.8 100.0

(%I

Mortality

II

963.8d 114.3

152.7 -c -

366.8” 24.2 -

km)

Final

weight

LATHYROGENIC WITH

-i+ -

+

-I-

-

i-t + + -

-

Spinal cord

+ + -

+

+

-

f + + -I-

-

+ + -

-

+

t + -

-

+ + -

-

Spinal cord

+

: -t-

+

Optic lobes

Lesions

THIOIJRACIL

changes

AND

Cerebellum

Nuclear

THYROXINE

+ + -

-

-

+ + -

-

Cerebellum

Demyelination

-t+ -

-

I_

+ + -

-

Optic lobes

NEUROLATHYRISM

IN

DUCKLINGS

445

Trial 4 Thiouracil, as 0.2% of the diet, depressed growth and increased the severity of the toxic effects of both INAH and SCH (Table II). Microscopic lesions in the CNS were the same with or without the addition of thiouracil to the test diets. Thiouracil alone induced no detectable lesions in the CNS. The thyroid glands were markedly enlarged in the ducks fed thiouracil. Weights for the thyroids of 10 ducks fed thiouracil averaged 0.4452 gm, whereas the average weight for the control groups was 0.07010 gm. Microscopically, the thiouracil-thyroids were hyperplastic. The follicles were reduced in size, lined by high-columnar epithelium, and contained little to no-stainable colloid. Piling up of the hyperplastic epithelium obliterated the lumen in many of the follicles. DISCUSSION Neurologic alterations, observed in rats fed semicarbazide, have been ascribed to be secondary to the osseous changes (Dasler, 1958). Cameron (1962) observed degeneration and necrosis of motor cells of the spinal cord of ducklings fed SCH; this seemed to indicate a direct neurotoxic effect of the drug. Data obtained in this study confirm the findings of Cameron, although our results differ somewhat from his. We were unable to confirm the presence of a low-grade inflammatory reaction in ducks fed semicarbazide. In addition, the marked nuclear alterations seen in our test ducks were not described by Cameron. Absence of gross skeletal deformities with cord compression supports the concept that semicarbazide has a direct neurotoxic effect, and the alterations in the cell bodies of motor neurons are not secondary to any osseous changes in the vertebral column. A direct neurotoxic effect of bis-P-(cyanoethyl)-amine was observed by Bachhuber et al. (1955), who reported hydropic degeneration or necrosis of the Purkinje cells in the cerebellum and ganglion cells of the anterior horn of the spinal cord. There were minimal to moderate osseous changes in their experimental rats. Although mortality and growth depression were more severe in the SCH-fed ducks, more marked microscopic lesions were present in the brain and spinal cord of INAHfed ducks. In addition to nuclear changes present in cerebellar Purkinje cells and motor neurons of the spinal cord of both test groups, demyelinating lesions were observed in the cerebellar medulla, the dorsal funiculus of the spinal cord, and the optic lobes. Demyelination was restricted to the INAH birds, Based on microscopic lesions, it appears that the mode of action of these two neurotoxic substances may be different. Also, it may be assumed that the demyelination in the INAH-fed birds cannot be ascribed to the nuclear alterations because these were observed in SCH-fed birds as well. The demyelination was apparently a direct response to INAH administration. In osteolathyrism there is a weakening of intermolecular collagen-bonding which results in an increase in the amount of salt-soluble collagen (Levene and Gross, 1959). Levene (1962) observed that the addition of certain carbonyl compounds to incubation media containing bones from chick embryo treated with INAH or BAPN diminished the amount of extractable collagen. The reversal of the INAH effect was complete with nn-glyceraldehyde and marked improvement was observed with salicylaldehyde. These 2 compounds had no demonstrable effect on the neural lesions induced

446

W.

W.

CARLTON,

C.

E.

HUNT,

AND

P.

M.

NEWBERNE

in our ducks by INAH and SCH. Both carbonyl-donating substances lowered weight gains. Tissue alterations referrable to feeding of the carbonyl-donors were not found in the brain or spinal cord. Selye (1958) observed that the syndrome of excitement, choreiform movements and circling induced in rats by aminodipropionitrile could be prevented by comparatively small doses of thyroxine. Thyroxine in a daily dosage of 0.25 mg per animal had a remarkable inhibitory effect on the development of aortic lathyrism in rats fed Lathyrus odoratus meal (Pyorala et al., 1959). Ponseti (1957) found that low doses of L-triiodothyronine suppressed the mild forms of aminoacetonitrite poisoning in rats, but extremely high doses of thyroxine were necessary to accomplish similar results. In our studies thyroxine was found to be extremely toxic to young ducklings and failed to prevent neural lesions induced by SCH and INAH. Thiouracil at the level used was not so toxic as thyroxine, although it reduced weight gains as compared to the control ducks. When thiouracil was added to the SCH and INAH diets, it caused a progressive increase in mortality throughout the experimental period; thiouracilsupplemented lathyrogenic groups did not grow as well as the groups which received SCH or INAH alone. We are unable to interpret the neurologic lesions observed in our experiments. Metabolic pathways of these compounds in ducklings are not known. Smith et al. (1963) have described cellular changes in the cortex, hippocampus, and cerebellum in rats which received small doses of cyanide. Lesions included neuronal degeneration and cell loss. It has been shown that hydroxyocobalamin readily takes up cyanide to form the harmless cyanocobalamin and can protect the experimental animal against lethal doses of the former (Mushett et aZ., 1952). Data are not available on the activity of vitamin B1:! against SCH and INAH, but Barnett et aZ. (1957) reported that the addition of 100 mg/kg of vitamin B 12 appeared to have no effect on the occurrence of leg deformity in turkey poults fed BAPN. The lathyrogenic drugs, SCH and INAH, may exert their influence through inhibition of enzyme systems. Monoamine oxidase inhibitors have been shown by Palmer and Noel (1963) to produce neurological lesions in dogs. Sodium diethyldithiocarbamate has been reported to inhibit monoamine oxidase (Yamada and Yasunobu, 1962) and Mur et al. (1956) reported a destruction of myelin in rabbits after repeated intravenous administration of a solution of diethyldithiocarbamate. SUMMARY White Pekin ducks fed 0.1% isonicotinic acid hydrazide (INAH) or 0.1% semicarbazide hydrochloride (SCH) showed, after about 2 weeks of feeding, signs of motor disturbances. These included tremors, ataxia and paresis. Microscopic lesions included degenerative changes in the nuclei of cerebellar Purkinje cells, motor neurons of the ventral horn of the spinal cord, and neurons of the medulla oblongata. An additional lesion in INAH-fed ducks was a non-inflammatory demyelination. It was most severe in the cerebellar medulla but was also observed in the dorsal funiculi of the spinal cord and in the optic lobes. Supplementation of the SCH diet with nr-glyceraldehyde (0.5%) or salicylaldehyde (0.5%) failed to prevent the neural lesions. m-Thyroxine was found to be extremely toxic and had no protective activity against SCH- or INAH-induced changes in the brain and spinal cord. Thiouracil as 0.2% of the diet did not prevent the lesions induced by SCH or INAH feedings and appeared to enhance their toxicity as there was greater weight reduction and increased mortality in thiouracil-supplemented groups.

NEUROLATHYRISM

IN

DUCKLINGS

447

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T. E.,

LALICH,

J. J,, ANGEVINE,

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P. M.

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H. (1958). Prevention of the ECC syndrome by thyroxin. J. Clin. Exptl. Psychopathol. 19, 97-101. (1963). Neuropathological changes in chronic SMITH, A. D. M., DUCXETT, S., and WATERS, A. H. cyanide intoxication. Nature 200, 179-181. STOCKMANN, R. (1917). Lathyrism in man. Edinburgh Med. J. 19, 297-307. WAWZONEK, S., PONSETI, I. V., SHEPARD, H. S., and WIEDENMANN, L. G. (1955). Epiphyseal plate lesions, degenerative arthritis and dissecting aneurysm of the aorta produced by aminonitrites. Science 121, 63-65. (1962). Monoamine oxidase. II. Copper, one of the prosthetic YA~~ADA, H., and YASUNOBU, K. T. groups of plasma monoamine oxidase. J. Biol. Chem. 237, 3077-3082.

SELYE,