Lathyrism has been produced in rats by manyworkers (@, ii, 13, 14, @0).The dominant signs ofthis disease in rats are kypho-scoliosis of the dorsalvertebrae, prominence of the sternum, bowing andwidening of the long bones, and hernias. The microscopic observation of delayed ossification withwidened epiphyseal lines and new formation ofsubperiosteal bone around the shaft has also beenreported (14, @0).When the active principle ofLathyrns odorata was made available to us incrystalline form,' we decided to study the effect ofthis material on the bones and cartilages of theembryonic salamander.

MATERIALS AND METHODS

All animaLs used were Amblystoma punctatum.Thirty embryos in the neural crest stages 16—18(harrison) and ten embryos in the early tail budstages @3—@5(Harrison) were placed in 1 mg. per

cent solutions of the crystalline Lathyrus factor inspring water. The animals were allowed to developto stage 46 ±(the feeding stage).

Gross observations were made daily. Animalswere sacrificed either at stage 46 ±or when theyfailed to feed; they were fixed in Bouin's fluid andserially sectioned at 10@ Sections were preparedin either the frontal, transverse or sagittal plane.Most sections were stained with hematoxylin andeosin; periodic acid Schiff, iron hematoxylin, tnchrome, and elastic tissue stains were also used onselected sections.

RESULTS

By stage 41, small tumorous growths had appeared on the dorsolateral aspects of the trunks

S Supported in part by a grant-in-aid from the United States

Navy, Office of Naval Research, Nonr 266(15), and in partby the Orthopedic Research Fund through Dr. Alan de ForestSmith.

t Fellow of the Damon Runyon Memorial Fund.1 From Dr. F. M. Strong of the University of Wisconsin

through the courtesy of Dr. Karl Meyer.

Received for publication December 11, 1954.

and/or tails of animals maintained in the Lathyrusfactor. At stage 46 ±, these animals were shorterthan the controls. In addition, the mandible wasshorter than normal; the forelimbs were distorted,and all Lathyrus-treated animals showed small tumors on the dorsolateral aspects of their trunksand/or tails, approximately 1 mm. in diameter(Fig. @).Few Lathyrus-treated animals survivedbeyond the feeding stage.

Mi.croscopu@description.—The tumefactions ohserved grossly were found to have resulted frommarked local overgrowths of the notochord. Theselarge notochordal masses burgeoned into the surrounding tissues, often presenting immediatelybeneath the epidermis (Figs. 3 and 4). They aroseat random points along the notochordal rod, haying been observed in loci from the basicranium tothe tail, without evident regional selectivity. Inany larva, the number of notochordal excrescencesrarely exceeded three. In most specimens the unaffected portions of the notochord maintained theirgeneral shape and structure with relatively littledistortion. In many instances, at the site of theecchordosis, there occurred an inflection of thenotochord toward the tumor mass, giving rise to aplication or kink on the opposite side of thenotochord (Fig. 6).

The excrescent masses, which formed relativelyextensive tumors, arose from any radial sector ofthe notochord, thrusting aside or compressing allsurrounding structures. The tumors were mostcommonly rounded but also assumed elongatedprolate shapes, presumably owing to the pressureof adjacent tissues.

Each outgrowth emerged from the notochordalong a relatively broad base, without pedunculation, and its substance was continuous with thenotochordal parenchyma (Fig. 10). The tumormasses were sometimes slightly lobate or irregular,being traversed and vaguely subdivided by irregular meandering septal strands (Fig. 8). Thesebands were of delicately fibnillar aspect and varying thickness; they often enclosed small lacunae of

184

Tumors of the Notochord of the Salamander,Amblystomapunctatum, Produced by Crystalline Lathyrus Factor*

BARNET M. LEVY AND GABRIEL C. GODMANt

(Department of Dental and Oral Surgery and the HistOChemistrij Reaearch Laboratory, Faculty of Medicine,Columbia University, New York, N.Y.)

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LEVY AND GoD1@@AN—Lathyrism and Tumors of Notochord in Salamanders 185

basophilic or amphophilic and carminophilic matenial, and nonvacuolated cells. They were in intimate relation with the chordal cell borders, merging indistinguishably with them and with penipheral remnants of true sheath. The impression isgained that these fibnillar septa which subdividethe tumor are formed, at least in part, of chordalcell “ectoplasm―or of fibrils elaborated by it (24,

The cells composing the tumor were for themost part identical with the differentiated physalifenous cells of the mature uninvolved notochord, except for a greater prominence of the cellmembrane or “ectoplasm―along the septa. Smaller“transitional― or fusiform cells without vesiculated cytoplasm, rare in the intact notochord, wereencountered in the tumors with greater frequencybut were never very numerous. The occasionalmitotic figures seen in the tumors were in such presumably germinal cells (Figs. 7 and 8). The mitosesand the obvious increase in cell numbers testify totheir essentially proliferative hyperplastic nature.

In some of the tumor masses, the cells were cornpressed by the pressure of surrounding structuresinto a pseudo-squamous pattern, with their longaxes at right angles to the direction of the outgrowth and parallel with the long axis of the larva.The adjacent tissues and the greatly distendedatrophied perichordal mesenchyrne formed a continent envelope for the tumors, which were alwaysthus circumscribed but never enclosed in a truenotochordal sheath. In urodeles the notochordalsheath proper is represented by a thick inner fibrillar lamella and an outer hyaline “elastic―membrane. This sheath was completely or largelyabsent about the tumors. The disposition of thesheaths was best studied in cross-section after thePAS stain, which colors the fibrillar sheath andinternal septa brilliant magenta (Fig. 8). As it extended out from the base of the tumor, the innerfibrillar lamella became homogeneous and poorlyoutlined, with diminished affinity for stains. Thesepta in the tumor often appeared to merge penipherally with the frayed-out remnants of thefibrillar sheath. Similarly, the external so-called“elastic―membrane seemed to disappear into thepenichordal skeletogenous mesenchyme. The absence of a true sheath about the tumors gave themthe appearance of proliferations prolapsed throughbreaks in the notochordal sheath.

Wherever the burgeoning tumor abutted, preexisting structures were displaced and/or cornpressed. The severity of the resulting pressureatrophy of any organ was proportional to its degree of fixity. Thus, the spinal cord within thespinal canal, encroached upon by dorsolaterally ex

tending tumor, suffered severe compression as itwas forced against the neural arches. The developing vertebral arches themselves were often merelydisplaced along a longitudinal axis without cornpression, but the immobile basisphenoid cartilageoverlying an anterior ecchordorna exhibited moderate pressure atrophy. The rnyotomes were usually invaded and occupied by the notochordal bur

geons, which were often surrounded by atrophicmuscle fibers. Wax reconstruction of the notochordof one of the animals is shown in Figure 10.

The cartilages of the limbs and head appearedin some cases to be somewhat foreshortened, but

exhibited no degenerative changes.

DISCUSSIONIn the several species of mammal known to be

susceptible to the toxic factor of Lathyrus seed,young growing individuals are most significantlyaffected. In the rat, in which the experimental disease has been most carefully studied (6, 7, 11, 18,19, 20), lesions of the skeleton, particularly atepiphyseal lines, and of large blood vessels wereoutstanding. In view of this distribution of lesions,it has been supposed that lathyrism affected themaintenance of some component of the connective

tissues, presumably chondroitin sulfate-containingground substance (19) . It therefore seemed of particular interest to study the action of this interesting toxin on morphogenesis in embryonic life, andincidentally to observe its hitherto undescribed effects on a poikilothermic vertebrate, Amblystomapunctatum.

The toxic principle of L. odorata seed, isolated incrystalline form (8, 9, 22) made it possible to estimate dosage. It has recently been reported thatthe active compound is @3-(-y-1-glutamyl)-aminoproprionitrile (28), in which the toxic propertiesappear to reside in the $-aminoproprionitrilegrouping (8). The sensitivity of amphibian larvaeto the toxin is noteworthy. Using this relativelysimple compound, Chang and Witschi (4) are saidto have observed articular lesions and/or looseningof rnetameric septi in Xenopus larvae after treatment with concentrations of 1 :500,000.

From descriptions of the skeletal, articular andligamentous deformities of rats and, in retrospect,from the experiences of Chang and Witschi (reported after completion of our experiments), itmight have been expected that the principal effectof the Lathyrus factor on Amblystorna larvaewould be manifested in the skeletogenous and supporting connective tissues. There were, indeed, deformities of the mandible and forelirnbs not readilyexplainable on microscopic examination.

The finding of large tumorous ecchordoses in all

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Cancer Research186

of the larvae exposed to the poison was unanticipated. In form and behavior these masses had thequalities of benign tumors. At the stage in whichthey were examined, there were no evidences ofmitotic activity in uninvolved regions of the notochord; nor could the proliferation be recognized asregenerative in character, owing to the paucity ofundifferentiated germinal elements (8, 17, 18).Possibly, as in the first stages of regeneration of thenotochord in certain other amphibian larvae (17),amitosis may be the principal mode of cell divisionin the formation of the ecchordoses.

The Lathyrus toxin is believed to act chiefly orexclusively on certain mesenchymal connective tissues. The derivation and histogenetic affinities ofamphibian notochordal tissue have not been entirely decided. It is said to be capable of cartilaginous metaplasia (18). The origin of its fibroussheath has also previously been in some doubt; ithad been held to be a secretory product of chordal“epithelium―(10, 26), or the result of condensation of a premesenchymal or mesenchymatous (16)perichordal network, or the product of the synergistic action between chordal cells and mesenchyrne (12, 18). The experiments of Mookerjee(15) have definitely established that the sheath of

the urodele notochord is formed by extrachordalmesenchymal cells, induced to this function bycontact with notochord.

To explain the occurrence of ecchordomas in thelarvae consequent on lathynism, it might be considered that the toxin exerts a selective primaryeffect on notochordal tissue, stimulating it to proliferation. While this is possible, it is unlike anyeffect of the toxin hitherto described. Since defectsin the notochordal sheath are invariably found atsites of tumefaction, it is also possible that somedisturbance in the structural integrity of the meso@@dermal fibrous sheath induced by Lathyrus toxinis a prior event in the pathogenesis of the chordomas.

Experimental chordoma-like lesions were firstproduced by Ribbert (@1), who showed that puncture of the intervertebral discs of the rabbit resalted at first in progressive nodular growth of theextruded[notochordal material of the nucleus pul

posus. Congdon (5) has repeated and extendedthese experiments and noted that the nodularmasses of chordal cells that developed followingpuncture of the nucleus pulposus through the dischad the character of true benign neoplasms. Hedrew attention to the exceptional nature of thestimulus to proliferation of chordal tissue in thenucleus pulposus, in which it appears that releasefrom constrictive pressure promotes marked pro..liferation.

An analogous situation might obtain in the amphibian notochord released from its surroundingsheath at some loci. Amputation, or excision, withresultant release of normal turgor, is a stimulus toregenerative proliferation and differentiation (@,3,17, 18) while obstruction by scar tissue or othermechanical impediments inhibits chordal proliferation (1, 18). The effect of sheath injury alone, in amore comparable experimental situation, could bestudied by effecting a simple puncture in the notochordal sheath of the larva.

It would be more in conformity with the knowneffects of Lathyrus toxin on mesodermal ligamentous, fascial, cartilaginous, and bony structures in all other species to postulate that focaldegenerative or aplastic changes of the chordalsheath are the primary lesions in Lathyrus-intoxicated larvae. These would be followed as a secondany event by ecchordoses or prolapses of the turgidchordal tissue with resultant excessive proliferation. Tempting as this hypothesis may be, however,the histologic evidence from our experiments is insufficient to confirm it, and it must be concludedthat we do not know whether the toxic factor ofL. odorata is a selective primary stimulus to tumorous proliferation of notochordal cells, orwhether it brings this effect about through precedent deleterious action on the notochordalsheath.

SUMMARYAmblystoma punctatum embryos reared in a 1

mg. per cent aqueous solution of the crystals ofLathyrus odorata developed tumors of the notochord. The histologic appearance of these tumorsis described. Two possible mechanisms of patho..

Gross pictures of 1athyrUS-treated animals.FIG. 1.—The animal on the left is a normal control, stage

46. The other two animals were reared in 1 mg. per cent crystalline lathyrus factor from Harrison stage 16.

Fio. 2.—Highermagnificationofa stage46, 1athYTUS-treatedanimal, placed in 1 mg. per cent crystalline lathyrus factorsince stage 16. Note the distorted mandible and forelimbs, as

well as the tumors in the tail region.

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2

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Low power photomicrographs.FIG. 3—Periodic acid Schiff preparation, showing the

gradual disappearance of the notochordal sheath as well as theinternal sept@@within the tumor. (Frontal plane).

FIG. 4.—Low powerphotomicrograph illustrating the tumorcompressing the spinal cord. The tumor extends into the adjacent myotome.

Fic. 5.—Low power photomicrograph of sagittal section ofembryo showing notochordal tumors.

FIG. 6.—Notochordal “kink,―sagittal section.

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FIG. 10.—Picture of wax reconstruction of notoehord aiid

tumors of one of the embryos (stage 46).

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LEVY AND GoD@w@r—Lathyrism and Tumors of Notochord in Salamanders 187

genesis of these lesions are discussed. The suggestion is made that these tumors arise as a consequence either of a focal degeneration of the notochord sheath, leading to secondary proliferativeecchordoses or prolapses, or of a direct stimulationto proliferation of the notochordal cells.

ACKNOWLEDGMENTS

The facilities of the amphibian embryological laboratory ofthe Department of Anatomy, as well as the helpful suggestionsof Drs. S. R. Detwiler and W. Copenhaver, are gratefully anknowledged.

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1955;15:184-187. Cancer Res   Barnet M. Levy and Gabriel C. Godman 

, Produced by Crystalline Lathyrus FactorpunctatumAmblystomaTumors of the Notochord of the Salamander,

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