Oral changes in experimental lathyrism (odoratism)

Research ORAL CHANGES

IN EXPERIMENTAL

LATHYRISM

(ODORATISM)

GEORGEA. KRIKOS, D.D.S.,* ALVIN L. MORRIS, D.D.S.,** WILLIA~Z S. IIAJIltoNn, B.A., AND HOWARDH. MCCLURE, JR., B.A., ROCHESTER,N.Y.

L

ATHYRISM is a disease which has been known since the time of Hippocrates. It has been described as affecting the central nervous system of human beings who have ingested large amounts of certain lathyrus peas, such as TJ. sativus, L. cicera, and L. clymenum, for a long period of time.ll 2 Many animals are susceptible to the above lathyrus species, but the rat is notoriously resistant. In 1933, however, Geiger, Steenbock, and Parsons” fed rats a diet containing seeds of Lathyrus odoratus, the common flowering sweet pea, and succeeded in producing a disease which principally affected the connective tissues. Fibrous connective tissue, cartilage, and bone exhibited profound pathologic changes. Geiger and associates also observed that the symptoms were more severe and appeared earlier in weanling than in adult rats. A similar syndrome can be elicited by feeding seeds of L. hirsutus,4 L. tingitanus,4 and L. pusillus.5 In 1954, Dupuy and Lee6 reported the isolation from lathyrus peas of a crystalline substance which, when fed to rats at the level df 0.75 to 1 per cent, produced the same lesions as the peas themselves. &hilling and Strong? characterized the substance as beta- (gamma-L-glutamyl) -aminopropionitrile and deLater it was found that the toxicity of betaveloped a method for its synthesis. (gamma-L-glutamyl) -aminopropionitrile lies in the beta-aminopropionitrile portion of the mo1ecule.8-1o This material has a simple chemical structure. It consists of a two-carbon chain having an amino group at one end and a nitrile group at the other. Ponseti and Shepard, I1 Robinson and Bast,12 Walker and Wirtschafter,l” Menzies and Mills,l’ and others,?, IF,have reported in some detail the gross and From the Department of Pathology and the Division of Dental Research, University of Rochester, School of Medicine and Dentistry. Presented at the thirty-fifth general meeting of the International Association of Dental Research, Atlantic City, New Jersey, March 21-24, 195’7. This study was aided in part by grants-in-aid from the National Institutes of Health, Bethesda, Maryland, Nos. GF-4779-C2 and DF-4871X2. *United States Public Health Service Postdoctorate Research Fellow of the National Institute of Dental Research. **United States Public Health Service Postdoctorate Research Fellow of the National Institute of Dental Research. Present address: School of Dentistry, University of Pennsylvania Philadelphia, Pennsylvania. 309

microscopic changes seen in t,his caondition. These have included skeletal dcformit,ies. hernias? aortic ~IIC:II~~NW, ancl par;~ iysis of the hindlimbs. The skelctal deformities have ocacurrtld in sc~~eral sitc‘s. ‘l’hc stcrnrlm bc>comes angulatcd and protrudes, giving the thorns a pig~ol~-bl*(~;~st aI,I>cal*all(dt?. The vertebral column develops a kyl)hoscoliosis involving t,he thoracic and lumbar vertebrae. There is a slipping of the epiphyses, cspeci&of the femur, tibia, and humerus. Subluxations and dislocations of the sl~ouldcr and diastasis of the sacroiliac joint are commonly seen in rats receivin g beta-aminopropionitrile. The epiphyseal plates are markedly enlarged and the costochondral junctions are prominent, so that in severe cases the!- simulate a rachit,ic rosary. In the areas of ligamentous and tendinous attachments the pcriostcum becomes greatly thickened, and large numbers of bony spiculcs arc formed. The periosteal new bone formation gives rise to exostoses which markedly disfigure the bones. Hernias are rarely seen in the normal rat. Jn lathprism, however, approximately 25 per cent of the experimental animals develop hernias, most of them in the dorsal region. Many of t,he animals also develop aortic aneurysms, a lesion not otherwise encountered in the rat. An early symptom of lathyrism is the development of a peculiar gait, characterized mainly by the maintenance of the hindlimbs Later, hindlimb paralysis may develop. in an extended position during walking. A careful analysis of the changes seen in lathyrism indicates that beta-aminopropionitrile exerts a specific effect upon connective tissue, particularly cartilage, Beta-aminopropionitrile may be utilized, bone, and fibrous connectirc tissue. therefore, as a tool for the study of the metabolism of these tissues, and in this respect it belongs in the same category with vitamin C and cortisone. An understanding of the diseases affecting the periodontal tissues presupposes an understanding of the metabolism of connective tissues. It is with this in mind that the study of the effect of beta-aminopropionitrilc on oral ronnectirc tissue was undertaken. Materials

and Methods

Seventy malt and female Wistar rats, 21 days old, were divided into control and experimental groups. There was an equal distribution of sexes hctween th(l two groups. The controls were Ped at1 libitum a synthetic diet of the following formula: 10 per cent crude casein (Borden’s), 20 per cent dried brewers’ yeast, 63 per cent Cerelosc, 4 per cent Wesson salt mixture, and 3 per cent olive oil. The diet also contained 1850 I.U. vitamin A acetate, 5.9 I.U. vitamin D, 10 mg. alphatocopherol, and 1.2 mg. vitamin K per kilogram of diet. Methionine at a level of 0.86 per cent was also added to the diet. The experimental animals received ad libitum the above diet to which 0.2 per cent beta-aminopropionitrile” was added. Fresh batches of diet were prepared every seven to ten days, and each time freshly distilled beta-aminopropionitrile was used. The diet was kept in brown glass jars in a refrigerator. The uneaten portion was emptied from the feed jars and replaced with fresh diet each day. of

*The Research,

beta-aminopropionitrile Abbott Laboratories.

was generously North Chicago,

supplied Illinois.

by

Dr.

Robert

D.

Coghill.

Director

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Control and experimental animals were sacrificed after three, six, nine, thirteen, seventeen, twenty-three, and twenty-eight days on the diet. On each of these days five control and five experimental animals were used. The oral cavity was examined grossly, and then one-half of the mandible was stripped of soft tissues for the observation of gross changes. The other half was fixed in Bouin’s fluid, decalcified in a mixture of’equal amounts of 45 per cent formic acid and 20 per cent sodium citrate, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. Observations All experimental animals exhibited some or all of the previously mentioned ehangcs. Gross examination of the gingivae and the remaining oral mucosa did not reveal any abnormalities. Notching at the incisal edge of the incisors of the experimental animals was not observed, in contrast to the findings of Dasler.16 At the beginning of the experiment the first two molars had already erupted. It was noted that the third molar erupted at the same time in experimental and control animals (thirty-fifth day after birth). The rats sacrificed on the sixth experimental day exhibited an increased mobility of their molar and incisor teeth. This mobility increased rapidly during the first two weeks of the experimental period, following which there was no further change. The mandible presented characteristic changes similar to those seen in other bones. The condyle, the coronoid process, and the angle of the mandible were enlarged and the oblique ridges on the external and internal surfaces were prominent. On the lower border of the body of the mandible in the area of the first molar a similar protrusion developed (Fig. 1). Upon removal of the overlying skin, these enlarged structures were found to have a smooth, grayish white, firm covering. Removal of this sheath revealed rough bony surfaces. It should be emphasized that these bony deformities are the areas of tendinous and ligamentous attachments. The mandibles of the experimental animals were a reddish blue, as compared to the light gray of the controls. There was no difference between experimental and control rats with respect to the length of the mandible. However, the mandibles of the experimental animals were thicker buccolingually. The above-mentioned deformities were first observed in the group of animals sacrificed on the sixth experimental day and appeared to progress very rapidly up to the end of the second week, after which little additional change could be notctl. The period of greatest change was between the sixth and the ninth day. Periodontal Membrane.--,\/licroscopic ~saminahion of the periodontal membrane of t,he experimental animals revealed a loss of the normal arrangement and appearance of the fibroblasts and the intercellular substance. The fibroblasts exhibited at least two important changes. First, the basophilia of their cytoplasm was increased and, second, in many areas they were arranged in

KRIKOS,

Fig. I.-Exotoses at tre ated with beta-aminopropionitrile of control animal.

MORRIS,

areas

Fig. 2. Pig. 2.- Periodontal membrane Fig. K-Periodontal membrane thi rteen days. Fine flbrils,, without of an anlorphous eosinophlhc material

of

HAMMOND,

AND

muscular attachment for twenty-eight days

0. s., 0. M., R 0. I’

MCCLURE

Mnrrh.

in mandible of (below) compared

Fig.

a

1958

rat 49-day-old with man dibk

3.

of a :i4-day-old normal rat. of a 34-day-old rat receiving beta-aminopropionitrilc definite orientation, are embedded in increased surrounded by highly basophilic, palisadinn

? for amc mnts flbrobl a sts.

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rows (palisades). The size of t,hc fibroblasts was somewhat increased, but t.here was uo significant, change? in their numbers. ~+equently, the cellula~r outline had it wa,vy appearanw. ‘l’he inberwllular fibers exhibited a loss of their coarse, fibrillar appearance ant1 caharacteristic orientation. Instead, they appcarcd to consist of very fine fibrils, which had no definite orientation and were embedded in increased amounts of an amorphous eosinophilic material surrounded by palisading fibroblasts (Figs. 2 and 3). Microscopic alterations of the periodontal membrane were evident in the first group sacrificed after only three days of feeding. In this group the changes were usually limited to the apical bhird or half of the periodontal membrane. With the increase in the length of the experimental period, the alterations became more severe and more widely distributed, affecting larger areas of the periodontal membrane, and reached a maximum in the rats sacrificed after thirteen days of feeding. Thereafter, the severity of the changes increased only slightly. All areas of the periodontal membrane were affected, with the exception of the bifurcation area which revealed only slight changes. The periodontal membrane of unerupted third molars with partially formed roots did not exhibit any pathologic alterations in the experimental rats sacrificed thirteen days after the initiation of feeding. Similarly, the periodontal membrane of erupted third molars was also devoid of pathologic changes, as shown in the experimental rats sacrificed after twenty-eight days on the diet. The rats of this latter group were 49 days old when sacrificed. In the experimental animals of all groups the gingival epithelium, the lamina propria, and the transseptal fibers of the periodontal membrane did not exhibit any changes. There was no difference between experimental and control animals in the position of the most apical point of the epithelial attachment, which in these young animals was located at the cementoenamel junction. Bone.-In the areas of tendinous and ligamentous insertions the periosteum was markedly thickened and very cellular. It consisted of large numbers of closely packed fibroblast-like cells having basophilic cytoplasm, dense nuclear membrane, coarse chromatin network, and a prominent nucleolus. Mitoses were frequent. The amount of intercellular material was relatively small, and in some cases the palisading arrangement of the cells, so well tlemonstrated in the periodontal membrane, could be seen (Figs. 4 and 5). Large amounts of bone were laid down by the thickened periosteum. In this way, in the areas of muscle attachment, exostoses were formed. In the groups sacrificed between the third and thirteenth days after the initiation of feeding, the exostoses usually appeared as irregular masses and strands of eosinophilic material within a mass of highly cellular connective tissue (Fig. at later dates the bone of the exostoses became 7). In the rats sacrificed organized into trabeculated bone which, however, did not acquire any great compactness. In some cases the periosteum was no longer thickened but

I.

Fig.

5.

Fig.

6.

days mass

‘ig. 4.-Periosteum ‘ig. 5.-Periosteum xhibiting intense ‘ig. B.-Striated i with centrally

and muscular insertion of a 34-day-old rat Abroblastic proliferation. muscle fibers in area of placed nuclei. Same rat

of a 34-day-old normal receiving beta-alninopropionitrile muscular as shown

insertion in Fig.

5.

appearing

rat. for as

Inultin

stern eated

ORAL

appea tonne eight

Fig. ‘I.-Exostosis wing as irregular ctive tissue. Fig. 8.-Exostosis days. The bone

CHilNC;ES

in a S4-day-old masses and

IN

EXPERIMENTAL

rat strands

in a 49-day-old is trabeculated ant1

Fig.

7.

Fig.

8.

LATHYRISM

receiving beta-i-llninoprovionitrile of eosinophilic material rat receiving covered by

beta-aminouropionitrile a thin periosteum.

within

for thirte a highly for

en da .YS eel !lul .ar tw, en1 :s-

316

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MORRIS,

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AND

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19.58

instead was of normal appearance (Fig. 8). Generally, the bone of the experimental animals had thinner trabeculae ancl larger medullary spaces than that of the controls. The number of osteoclasts and the number and size of the ostcoblasts were increased in the c>xperimental rats sacrificed between the third and thirteenth days after the initiation of feeding. In the groups sacrificctl at later dates these differences could not be noted. In the vicinity of the thickened periosteum, the connective tissue SIXI’rounding the striated muscle fibers exhibited an increased cellularity similar to that seen in the thickened periostcum. The muscle fibers near the pcriosteum often appeared as multinuclcatad masseswith centrally placed nuclei (Fig. 6). The striated muscle fibers located at a distance from the arra oi insertion did not reveal any abnormalities.

Condylar Cartilage.-The first changes WPIY seen in the expcrirncntal rats sacrificed on the thirtl cxptrimcntal day. Tn thcsc animals the changes did not involve the entire thickness of the cartilage to an equal degree but were more pronounced in the metaphyseal third. Tn this area the chondrocytcs were basophilic and exhibited a dccrcased cytoplasmic and nuclear vacuolization. In the experimental rats sacrificed after six days on the diet, the alterations were again more pronounced in the metaphyseal third and consisted of the presence of chondrocytes arranged in isogenous groups, the appcara.ncc’ of which will hc described in greater detail brlow. With increase in length of the experimental period, the entire thickness of the cartilage prcscntcd changes until finally in the most aclvanced stagrs th(a condylar cartilage appeared enlarged and covered by a thick and highly cellular perichondrium. The cartilagc cells were haphazardly arranged. The nonuniformity of chondrocytes. which is characteristic of normal cartilagca l)a.rticipating in endochondral ossification, was also lost and the cartilage cells assumed :L uniform appearance throughout the whole thickness of the cartilage. The cells were small and devoid of cptoplasmic or nuclear vacuoles. Their cytoplasm was intensely basophilic and their nuclei exhibited a coarse chromatin network. They wert arranged in isogenous groups of two to four cells each. Within a group the cells were separated from one another by thin septa of matrix, and the cells of one group were separated from those of another by larger amounts of matrix. These cells, arranged in isogcnous groups, were the prevalent cell form in the condylar cartilage and wcrc present throughout its entire thickmu. Scattered among these cells were a few normal-appearing cells at variOLIS stages of maturation. The cartilage matrix revealed foci outlined by irregular borders and containing some granular material. Generally speaking, the cartilage was thicker and the cells were smaller ancl more numerous. Wecause of the smallness of the cells, however, there was also an apparent increase in the amount of cartilage matrix per unit area. The metaphyseal border of the condylar cartilage had an irregular outline and lacked the scalloped appearance of the normal cartilage. The number of lacunae opened by the vascular tufts from the acljacent hone narrow was limited. In some

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casles a thin transverse lamina of bone was deposited on parts of the metaph LYSeal border. The bony trabeculae were fewer, thicker, and more irregu liar in 1;heir orientation. No abnormal amounts of osteoid appeared to be deposi ted (Fi gs. 9 and 10). All of the above changes did not necessarily affect the entire Fig.

Fig.

twe 3LIY

Fig. Y.-Condylar cartilage Fig. lO.-Condglar cartilage nty-eight days. The matrix arranged in isogenous groups.

Y.

10.

of

a 4Y-(lay-old normal rat. of a 4Y-day-old rat receiving The chondrocytes is disrupted. and do not exhibit vacuolization.

beta-arninoyropionitrile show a unifornl

appeara

fo1 rice,

318

lil%IKOS,

MORIXIS,

HAMMOND,

AND

MC: CLUEE

0. s.. 0. M., & 0. P. March,

1958

condylar cartilage. In some cases a longitudinal segment of the cartilage and its adjacent bony trabeculae were normal in appcarancc, whereas the remaining part rcvea~lcd all of the changes described.

Discussion The microscopic observations indicate that the pathologic changes of the periodontal membrane in lathyristn concern both the fibroblast and the’ intercellular substance. It could be expected that the described changes in the periodont,al membrane would result in a rctluction of the efficiency with which the toot,h is anchored in its socket and thus offer an explanat,ion for the incrcascd tooth mobility which was observed grossly. Dasler’s17 suggestion that the periodontal mcmbranc may bc altered, based upon the cas(’ with which incisors could bc extracted in experimental rats, has been con firmed. It is interesting that, although the periodontal mcmbranc exhibited unquestionable pathologic changes, the lamina propria of the gingivae had :I normal appearance. It is possible that the differrnce in the response of these two tissue to beta-aminopropionitrilc may be dne to the fact that the periodon tal membrane is subject to a grcatcr mechanical stress than the gingivac. Support for this hypothesis can bc found in the fact that the thickening of th(’ periosteum and the periostcal new bone formation arc confined to arcas of muscular and ligamentous attachments and that transection of the spinal cord reduces the skeletal changes in the lower extremitics.18 The possibility also exists that the difference in response may be the result of the higher growth rate of the periodontal membrane. There is evidence suggesting that the susceptibility to compounds with lathyrous activity is proportional to the rate of growth. For the same experimental period, the severity of lesions is mulls greater in weanling than in adult rats”, I1 although Selye’” has raised a qucstion in this regard. Rat embryos are wry susceptible to beta-aminopropionitrile and are resorbed if the feeding of the maternal adult is extended beyond the sixteenth day of gestation.“’ Beta-aminopropionitrile in minute concentrations (0.5 microlitcrs per liter) produces pronounced changes of the mesodermal tissues of Xenopus larvae.21 Growth hormone intensifies the severit? of the skeletal changes in lathyrism.22 Finally, the suggestion is offered that the different response in gingivae and perioclontal membrane may rcfiect metabolic differences in the two tissues. Our observations on the periodontal membrane of the third molars of the experimental animals may help elucidate the diffcrcnce in the rcsponsc of the gingival lamina propria and the periodontal membrane to beta-aminopropionitrile. It is known t,hat the growth rate of the periodontal membrane is high, especially in young animals during the period of root formation and eruption. It is also believed that the> periodontal m~wtl~ratte of unerupted teeth is not subjected to any significant mechanical stress. Our observation that the pcriodontal membrane of unerupted third molars is free of changes suggests that mechanical stress is import,ant in t,he a,ltcrations in lathyrism. However, thcl

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periodontal membrane of erupted third molars (4%clay-old experimental rats sacrificed after twent,y-eight days on t,he diet) was also devoid of pathologic changes. It may bc mrntioned that, according to &hour and RIassler,23 the third molars of rats erupt on the thirty-fifth day and are in functional occlusion on the fortieth day after birth. A delay in the eruption of the third molars of the cxperiment,al animals was not observed, and this eliminates the possibility that before sacrifice they had been in functional occlusion for only a short period of time. However, although concrete evidence for this is lacking, it is possible that the third molars of the experimental animald may not have been in functional occlusion at all. The changes in the condyle and the malposition of the teeth due to the bony deformities may have kept the third molars out of occlusion. These observations seem to emphasize the importance of mechanical stress in the development of the morphologic changes characteristic of lathyrism. It must be pointed out that, on the basis of function alone, one would expect alterations of the transseptal fibers. Lack of changes in these fibers may merely reflect a smaller functional demand than that experienced by the remaining fibers of the periodontal inembrane. From the microscopic observations, some of the deviations from t,he normal process of endochondral ossification may be deduced. The absence of cytoplasmic and nuclear vacuolization suggests interference with the maturation of the chondrocytes. The presence of isogenous groups of chondrocytes distributed throughout the thickness of the cartilage suggests that proliferation of chondrocytes occurs throughout the cart,ilage rather than only adjacent to the perichondrium. The cellular and matrix changes of the cartilage are associated with an inhibition of the opening of the lacunae and suggest a decreased rate of endochondral ossification. The etiology of the intense fibroblastic proliferation of the periosteum and the formation by it of large amounts of bone is unknown. Both these changes account for the deformities of the bones of the experimental rats, and the thickened periosteum provides the deformed areas with a smooth, grayish whit,e, firm covering. The decreased thickness of the trabeculae and the enlargement of the medullary spaces may explain the observation that the mandibles of the experimental animals arc easily fractured and have a red, congested appearance. The changes of the striated muscle fibers in the vicinity of the thickened periosteum are similar to the changes seen during muscle regeneration. In lathyrism the rate of progress of the gross and microscopic pathologic changes is not constant. The changes usually reach a maximum in nine to thirteen days, after which the increase in severity is minimal. In the case of the periosteum the change may even regress, as shown by the presence of a large exostosis covered by a normal-appearing periosteum. Changes were observed as early as three days after the initiation of feeding. The total amount of food ingested during this period is approximately 20 grams, and consequently the intake of beta-aminopropionitrile during the

320

KRIKOS,

MORRlS,

HAMMOND,

ANT)

MC (:I,URE:

0. s 0. nr., & 0. I’. March. 195s

Silfllc periotl ot’ tilrltb is il]~~~l'O~illl;ltc~l~ otlly 0.04 grylul. This r~mphasizcs t,htl t’xt.rc’llt(’ clflgtSc~t~of’ 1cosicaity 01’ l~t~t;l.-i~t,linol,l~ol)io~litI~ilc tc, thtb nlt~t;~l>olislll of the’ (*cmtlt~ctiv(~ tisslic, of the f*:11. The ch(~micn.1 tIatut~(~ 01’ thcb conllcc+ivcb tissue changct antI the mcxtabolic site at which hclta-anlinoprol>ionitrilc acts arc, at present, very poorly understood. Currently, thr consensus is that in lathyrism it is not the calcium and phosphorus metabolism that is at fault,“, Ii. s but rather it is the organic component of the connective tissuas that. is primarily affected, perhaps the metabolism of chondroitin sulfate.“. 2j Attempts bo determine the metabolic site of action of beta-aminopropionitrile by administering chemical compounds having a chemical structure similar to that of beta-aminopropionitrile,8V1”, “’ by injecting isotopically labcllcd hcta-aminopropionitrile,“7 or by attempting to discover compounds that may hare a protcctivc or aggravating action on the developmt>nt of changes seen in IathyrismR~ I99 PC.28-31have thus far failed to clarify the problem.

Summary I. The oral tissues of wcanling malt and female rats fed a diet containing 0.2 per cent bct,a-an2inopropionit~,ilc cxhibitcd gross and microscopic changes. In the regions of trndinous attachments the mandible developctl cxostoses charactcrizcd by thick, highly cellular pcriostcum r:overing bone composed of thin trabeculac and broad marrow spaces. The mobility of the teeth was increased. The fibroblasts of the periodontal membrane exhibited incrcascd cytoplasmic basophilia and palisading. The collagen fibers were fine, tlisorientcd, and embedded in an increased amount of an amorphous eosinophilic material. The condylar cartilage was cnlargcd. The matrix presented foci having The cartilage cells an irregular outline and containing granular material. were arranged in isogenous groups and failctl to undergo enlargement and vacuolization. 2. Beta-aminopropionitrile appears to have a specific effect upon the connective tissues of the rat. Cartilage, bone, and fibrous connective tissue arc affected. The changes involve both the cells and the intercellular substance. 3. The observations on the periodontal membrane suggest that mechanical stress may be important in the dcvclopmcnt of the morphologic changes characteristic of lathyrism. 4. Beta-aminopropionitrile appc’ars to bc a useful tool for the stutly of the metabolism of connective tissues. taking

The authors are indebted to Dr. Roger the microphotographs presented in this

Terry article.

of

the

Department

of

Pathology

for

References 1. Stockman, R.: Lathyrism, J. Pharmacol. & Expel. Therap. 37: 43, 1928. 2. Young, T. C. M.: A Field Study of Lathyrism, Indian J. M. Research 15: 453, lUz?j. 3. Geiger, B. J., Steenbock, H., and Parsons, H. T.: Lathyrism in the Rat, J. Nutritioll 6: 427, 1933.

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