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REVIEW ARTICLE CONGENITAL SKELETAL MALFORMATIONS AND CLEFT PALATE INDUCED IN GOATS BY INGESTION OF LUPINUS, CONIUM AND NICOTIANA SPECIES K . E . PAIVTER,'
R.
F . KEELER,' T . D .
Bulvcx2 and R. J.
CALLANZ
'USDA/ARS/Poisonous Plant Research Laboratory, 1150 East 1400 North, Logen, UT 84321, and Animal Dairy and Veterinary Science Department, Utah State University, Logen, UT 84322, U.S .A. (Receivedjorpublication 11 1Nay 1990)
R. F . KEELER, T . D . Burrcx and R. J. CALLAN . Review article - Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupines, Corium and Nicotiana species . Toxicon 28, 1377-1385, 1990 .-Three piperidine alkaloid containing plants, Coniem maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupines formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities . Clinical signs of toxicity included those reported in sheep, cattlé and pigs-ataxia, incoordination, muscular weakness, prostration and death . One quinolizidine alkaloid containing plant, Lepinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring. K . E. PANT>~,
INTRODUCTION
Corium macelattan (poison-hemlock), Nicotiana glauca (tree tobacco), Lupines caudatus (tailcup lupine) and Lupines formosus (lunara lupine) have been used to experimentally induce crooked calf disease in cattle (KEELEIt, 1974; KEELER and BALLS, 1978; KEELER, 1979; SHLJPE et al., 1967a,b ; ICEELint et al., 1977; KEEL.E R and PANTER, 1989) . Lupine has also been associated with cleft palate in cattle (ICE~.ER and PANTER, 1989; SxvPE et al., 1968). In addition, Coniem and Nicotiana have induced multiple congenital contracture type skeletal malformations and cleft palate in pigs (KEELER and CROWS, 1983; IC>~LER et al., 1984; PANTER et al., 19ß5a,6) . Research has shown that feeding fresh Corium maculatum to pregnant sheep during 300 days of the gestation period induced transient multiple congenital contractures (MCC) which resolved spontaneously (PANTER et al., 198ßa). They subsequently determined that ConitWn reduced fetal movement in utero (PANTER et al., 19ß8b). Reduction of fetal movement may be a general physiological mechanism for induced MCC in animals and humans according to some workers (SWINYARD and BLECR, 1985; HALL, 1985; BANI{ER, 1985; HAGEetAN and WILLEMSE, 1987) . 1377
137 8
K . E . PANTER
~CH,-CH=-CH, CoIIlhs
Asabaslso
et at.
~CH,-CH,-CH, Y-C~~IeN~o
A~slodsdrloo
FIG . l . FOUR PII~ERIDINE ALKALOIDS PRESENT IN POISONOUS PLANTS KNOWN TO CAUSE CLEFT PALATE Arro MCC rrPE sKELErAL DEPEGTS .
Conüne and y-coniceine arc principal alkaloids in Conium . anabasine in Nicotiana gtauca and ammodendrine in Lapinas jormosus .
Many alkaloids in plants have been implicated as teratogenic agents . Conium maculacontains eight known simple piperidine alkaloids, two of which (conüne and gamma coniceine, Fig. 1) appear to be teratogenic in cattle, pigs and sheep (KEELER, 1974; ICEELER et al., 1980 ; PANTER, Ph.D . Thesis, University of Illinois, 1983) . In Nicotiana glauca the teratogenic agent is a pyridino-piperidine alkaloid, anabasine (Fig. 1) (KEELER, 1979 ; KEELER et al., 1984) . This alkaloid is also present in other plants, including Nicotiana tabacum (burley tobacco) (KEELER et al., 1984) . In lupine, the quinolizidine alkaloid anagyrine is apparently responsible for inducing deformities in cattle (ICEELER, 1976) . KEELER and PANTER (1989) demonstrated that another lupine, Lupines formosus, was teratogenic in cattle and contained piperidine alkaloids whose structures were similar to those in Conium and Nicotiana. The principal alkaloid in Lupines formosus, ammodendrine (Fig. 1) (Frrcx er al., 1974), is believed to be the principle teratogen (ICEELER and tum
PANTER, 1989) . KEEi.ER and BALLS (1978)
orally administered piperidine analogs of conüne to cattle . Results suggested these compounds must possess certain structural characteristics to be teratogenic; e.g. an alpha-substituted piperidine ring that must be saturated or perhaps contain a single double bond, and the alpha side chain must contain at least three carbon atoms. Many of the piperidine alkaloids now known or believed to be teratogenic meet these structural requirements . Livestock vary in their susceptibility to Conium, Nicotiana and Lupines teratogens. KEELER and BALLS (1978) and PANTER et al. (1988c) have shown that the piperidine alkaloids in Conium are most toxic to cattle, and less so to sheep, horses and pigs in that order and are most teratogenic to cattle, and less so to pigs and sheep in that order. Sheep are somewhat resistant to the toxic and teratogenic effects of Conium, Nicotiana and Lupines. Inherent metabolic and pharmacokinetic differences no doubt influence susceptibility among livestock species. An understanding of the variability may help elucidate the nature of poisoning and determine whether metabolism can be altered to reduce incidence, and identify the mechanism of teratogenicity .
Poisonous Plant Induced Malformations
1379
Teratogenic effects in susceptible animal species are similar and include MCC resulting in fixed extension or flexure of the carpal, tarsal or fetlock joints, enlargement of these joints, lateral deviation of the forelimbs, scoliosis, torticollis, lordosis and cleft palate . We report here experiments to determine the relative toxicity and teratogenicity in goats of Conium maculatum, Nicotiana glauca, Lupimis josmosus and Lupinus caudatus . The usefulness of goats as an alternate animal model for the study of mechanism of action of plant teratogens is considered . MATERIALS AND METHODS The estrous cycles in Spanish-type female goats weighing between 27 kg and 51 kg were synchronized using intravaginal peasaries' containing 45 mg of fluorogestone acetate. Twenty-four to 48 hours after sponge removal all female goats were bred by bucks of e simiL~r type. The female goats were randomly divided into the following five groups : Group 1, six goats, throe gavaged with Coniwn maculation seed (group la) collected in the fall of 1981 in Logan, UT, and stored at 4°C, and three gavaged with fresh Conium maculation (fall regrowth) (group 1b) collected fresh in the same location . In Group 2, 10 goats were gavaged with Nicotimra glauca collected in 1983 near Wickiup, AZ . Group 3 wnsistod of four goats gavaged with Lupinusjormosiu collected at Rio Vista, CA . Group 4 consisted of seven goats gavaged with Luplnur caudaticr collected in 1982 on the Session Mountains east of Woodrufl; UT. Group 5 (wntrol) consisted of nine goats gavaged with water only. All treated goats (Groups 1~) were gavaged with a known amount of plant material suspended in 1-31 of water twice daily for 30 days (30-60 days of gestation). Dosages (Table 1) were selected to assure clinical signs of toxicity in the dams, or were the maximum volume that could be successfully gavagecí . All goats had ad libitum accevs to water, trace mineral salt and alfalfa hay. Clinical signs of toxicity were observed and rewrded . At parturition, teratogenic effects in offspring wen assessed by two scientists, one of whom was unaware of treatment regimes in order to eliminate bias. Photographs and X-rays wen taken of severely deformed fetuses (Figs 2~) . RESULTS
Group 1--Conium maculatuur Clinical signs of maternal toxicity were modest to severe in goats gavaged with seed or fresh Conium maculatum, depending on the dosage . The most severe clinical signs occurred about 15 min to 2 hr after treatment and muscular weakness appeared to persist until the next dosage period . Signs of toxicity included trembling, muscular weakness and fasciculations, depression, ataxia and collapse. Clinical signs of toxicity were the same for seed and fresh plant. After the initial phase of the toxicity (about 2 hr) the goats' appetites were good and they often ate in a sternal recumbent position when hay was offered. They readily moved about but could stand for only X10 min early in the daily expression of toxicoses but could stand longer after several hours. More plant material was required to maintain the same clinical effect as the days passed during the experiment, suggesting ruminal or hepatic adaptation might be occurring. All clinical signs of toxicity in dams disappeared 248 hr after final treatment. The single goat fed Conium seed had triplets ; all had bilateral cleft palate (Fig. 2) and severe MCC (Figs 3,4). All were dead at birth. All kids had variable degrees of the following malformation expressions: fixed carpal flexure and rotation, elbow and shoulder joint rigidity, fixed and rigid stifle joints, severely flexed hock joints, rib cage deformities, sternal deviation, overextended fetlocks in the forelimbs, lateral rotation or rigidity of 'Pre-prepared pessaries were purchased from Intervet, B. P. 235-45 Avenue Joxi, 49002 Angers Cedex, France. Mention of a proprietary product does not wnstitute a guarantce or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
1380
K. E. PANTER et al.
F~a . 2 . Bilateral cleft palate induced by Conium aced, N. glauca and L . jormosus .
hock, stifle, and pastern joints in the rear limbs, and deviated spinal column with scoliosis, torticollis or lordosis . One of two goats gavaged with fall growth Conium had twins with variations of modest carpal flexure and elbow joint rigidity that were otherwise normal. The other goat had twins, one of which had equivocal carpal flexure and the other had modest carpal flexure and modest elbow rigidity, but appeared to be normal otherwise. Seed and fresh plant induced the same signs of toxicity, but signs persisted longer in the goat gavaged with seed . The persistence of the clinical signs appeared to parallel the severity of the malformations. Group 2-Nicotiana glauca Clinical signs of toxicity in ten goats gavaged Nicotiana glauca were moderate to severe and persisted between doses of plant material. The first signs of toxicity were evident about 15 min after treatment and were most severe by 1-2 hr after treatment. Signs of toxicity included muscular weakness in the legs and neck, ataxia, incoordination, muscular fasciculations, collapse and sternal recumbency. All but one of the 16 kids born to these goats had bilateral cleft palates and varying degrees of MCC type skeletal malformations. The malformations were the same as those described for Conium seed and are illustrated in Figs 2~. The severity of the malformations paralleled the severity of the clinical signs of toxicity in the dam. Group 3-Lupinus formosus Clinical signs of toxicity were slight to severe in four goats gavaged Lupinus formosus and were similar to those described for Conium maculatum and Nicotiana glauca, however, because of plant volume we were unable to increase dosages in some of the goats to
Poisonous Plant Induced Malformations
FIG. 3. OVEIiEICIENSION AT THE PASTIItN JOINTS, SEVERE FD® CARPAL FLEXURE, RIH CAGE DEPRESSION AND SPINAL COLUI~i DEPRL~ON (LORDOSiS) (ARROWS) TYPICAL IN RIDS FED Conitan SEED, N . giQüCO AND L . forIltOS7L4 .
Severity and location of malformation varied from kid to kid.
FIG . 4. PHOTOGRAPH of AN X-RAY OF A Rm IN A VENTRAL DORSAL VQ;W WITH THE HEAD EXTENDED .
Note the deviation and the severe curvature of the thoracic region, the wedging of the vertebrae and the abnormal rib and rib ar8e development .
138 1
1382
K . E . PANTER et al.
TABLe 1 . TOXICITY AND TIIüTOGEPIIC RE4IJL1'3 OF I~DING Conium maculatum, Nicotiana AND Lupinus CauL%atuS TO PItFX3NANT GOATS
No. of goats (mean wt, kg)
I (37) 2 (36) 10 (36) 4 (43) 7 (40) 9 (42)
Treatment
C. maculatuur (seed) C. maculatuur (fresh) N. glauca L. jormosus L. caudatus Control
Clinical toxicoses
Moderate to severe Moderate to severe Moderate to severe Slight to severe None to slight -
'Mean daily dosage g
130 387 200 530 500 -
glauca, Lapinasjormosas
Teratogenic results tNo . CP
$No. MCC
3/3 0/4 15/16 §3/9 0/14 0/20
3/3 3/4 15/16 4/9 0/14 0/20
'Daily dosage was the total of the a .m . and p .m. dosages . The dosage of fresh plant was adjusted daily due to variability in toxicity of material . tCP = cleft palate, all were bilateral cleft except 1 kid in the L . jormosus which was a unilateral cleft . $MCC = multiple congenital contractures and indicates the number of kids with MCC per number of kids born. §One goat died on day 36 of gestation, she had triplets but palate or skeletal defects were not discernible because of age and size of the fetuses . We would not expect closed palates until after 38 days gestation .
maximize clinical signs of toxicity (Table 1). Two of four goats had deformed offspring . Two kids from the same dam had bilateral cleft palates. One of two kids from another dam had a unilateral cleft palate, but the palate of its twin was normal . The skeletal defects were severe and similar to those described for Conium and N. glauca in the two kids with bilateral cleft palate . Both twins, one of which had the unilateral cleft palate, had moderate carpal flexure, but no other skeletal anomalies. The severity of the clinical toxicoses in dams paralleled the severity of the subsequent malformations in the fetuses. Group 4-Lupinus caudatus
Only slight clinical signs of toxicity appeared in two of seven pregnant goats gavaged
Lupinus caudatus. They exhibited mild muscular weakness in the cervical region and
carried their heads lower than other goats exhibiting the early stages of toxicity . These signs were evident only during the first few days of treatment, and did not persist between doses. These seven goats had 14 kids and all were normal with no skeletal or palate defects. Group S~ontrols
All nine control goats had normal kids (20 total) without cleft palate or skeletal abnormalities. DISCUSSION
Conium maculatum, Nicotiana glauca and Lupinus jormosus, all of which contain piperidine alkaloids, induced congenital birth defects in pregnant goats when gavaged during gestation days 30-60. The birth defects included cleft palate and MCC that were expressed directly or indirectly as flexure of the joints, torticollis, scoliosis, lordosis, rib cage depressions and wedging of vertebrae. The quinolizidine alkaloid-containing plant
Poisonous Plant Induced Malformations
1383
Lupines caudatus, however, did not induce any malformation in offspring when goats were gavaged during the same time period . The results showed that Conium maculatum, Nicotiana glauca, and Lupines formosus induce cleft palate and MCC in goats similar to deformities reported in cows and pigs suggesting that goats may be a useful alternate animal model to study plant teratogens and mechanisms of fetal effects. Our results presented here and elsewhere (KEELER and Pnrr~rm, 1989; Pnrr~t~It et al., 1990) suggest that livestock species either differ in metabolism of or in absorption and transfer of these plant toxins to the fetus. The three plants containing piperidine alkaloids, Conium, Nicotiana, and Lupinesformosus, induced cleft palate and MCC in the goats, but Lupines caudatus, which contains quinolizidine alkaloids did not cause significant maternal toxicity or teratogenicity in the goat. These and other findings suggest that piperidine alkaloids may be teratogenic in both species (cow and goat) but that the quinolizidine alkaloid anagyrine is teratogenic only in the cow. This species difference is interesting and suggests possible ruminal or liver metabolic processing differences. ICEELER and BnLIs (1978) proposed that certain minimum structural characteristics of piperidine alkaloids were required for teratogenicity in cattle . The structural requirements included an alphasubstituted piperidine ring whose alpha side chain was at least three carbons long. IC>~r m and Pntv~It (1989) hypothesized that, in the cow, minor metabolic changes in the quinolizidine alkaloid anagyrine from lupine could result in a piperidine alkaloid that meets the structural requirements for teratogenic piperidines. Thus, a piperidine alkaloid meeting these structural requirements may be the proximal teratogenic compound even when the quinolizidine, anagyrine is the suspected agent in the plant. This might explain why terata are absent in goats fed the anagyrine containing Lupines caudatus even though the plant causes cleft palate and MCC in cattle . Goats may not metabolically modify anagyrine in the rumen to a teratogenic piperidine as cows hypothetically do. Further research is needed to elucidate this possibility. The present results showed that MCC may occur alone or with cleft palate depending on the stage of pregnancy when exposure occurs . In animals and humans, the biological mechanisms) causing cleft palate and MCC type skeletal abnormalities are not fully elucidated. Cleft palate induction may involve the same biological mechanism as MCC induction even though the insult occurs at a different gestational period (see below) . MCC and cleft palate were here associated with plant toxins administered at different gestational periods. Pnrr~rEtt et al. (1985a) showed that only cleft palate occurred when swine were fed Conium maculatum during days 305 of gestation, and that feeding this plant during days 43-53 of gestation did not cause cleft palate but did cause MCC type limb and spinal malformations and associated rib cage abnormalities (PAIVTER et al., 19856) . When the same plant was fed from days 511 of gestation, the limb defects were less severe and rear limbs were effected more often then the forelimbs. As expected, (WILSbN, 1973; Sl~nxn, 1976) stage of gestation when exposure occurred determined terata expression. A correlation between a reduction in fetal movement and associated MCC and cleft palate by these plants and the alkaloids therefrom has been demonstrated (PAIVT'ER et al., 1988b; PANTER et al., 1990). In 1988, Pnrr~It et al. reported that Conium maculatum inhibited fetal movement in sheep for about 5-6 hr after each dose, before returning to normal by 12-16 hr. Fetuses born to ewes so dosed for several days showed modest carpal flexure, which resolved spontaneously by 8 weeks, and no cleft palate . We believed that the lack of severe birth defects (MCC and cleft palate) in those sheep resulted from intermittent rather than constant inhibition of fetal movement during the critical gestational period. Subsequently, PnN~It et al. (1990), and as reported here, showed that prolonged
1384
K. E. PANTER et al.
inhibition of fetal movement induced by Corium maculattim and Nicotiana glauca during critical stages of gestation resulted in cleft palate and MCC. Thus, duration and severity of fetal movement inhibition are directly correlated with deformity expression severity suggesting a mechanistic association. The relative roles of the primary pathologic condition or biochemical lesion and loss of fetal movement in the expression of MCC and cleft palate are not known. Further research is needed to clarify the teratogenic role of chemicals which could affect fetal movement at a critical stage of development but not cause primary pathologic changes. The goat is a good animal model for monitoring fetal movement with ultrasound because of size similarity to humans and because goats are docile. The association between loss of fetal movement and plant-induced birth defects must be clarified to be sure that the loss of movement is not independent of malformations and that these malformations do not occur with normal fetal movement . We suggest in the present experiment that the MCC resulted from loss of fetal movement induced by the CNS depressing effect of these plant alkaloids. We also suggest that the cleft palate was a direct result of mechanical obstruction of the tongue resulting from the same CNS depressing effect and lack of fetal activity . Acknow/edgements~ontribution of the USDA/ARS Poisonous Plant Research Laboratory in cooperation with the Utah Agricultural Experiment Station, Utah State University, Logan, UT 84322. Approved as Journal paper no . 3822 . The authors wish to thank LBw Det.[. Bat.ts, Tort Buctc, A~ Mac~ut,ts and Lt7w KEtso for animal care and assistance and Ms Tt~ WIERENGA for technical assistance . REFERENCES Barrxex, B. Q. (1985) Neuropathologic aspects of arthrogryposis multiplex congenital . Clip. Orthop . 194, 303. Frrctt, W. L., DOLINGI~t, P. M. and Drnn~eci, C. (1974) Alkaloid studies. LXVIII . Novel piperidyl alkaloids from Lupinesformosus. J. Org. Chem . 39, 2974-2979. HAGEMAN, G. and Wtt i a..aF, J. (1987) The pathogenesis of fetal hypokinesia. A neurological study of 75 cases of congenital contractur~es with emphasis on cerebral lesions. Neuropediarrics 18, 22-33 . HALL, J. G. (1985) Genetic aspects of arthrogryposis . Clip . Orthop . 194, 44-53. Kt~t~x, R. F. (1974) Conüne, teratogenic principal from Corium maculatum producing congenital malformations in calves. Clip. Toxicol. 7, 19206 . KEet .ea, R. F. (1976) Lupin alkaloids from teratogenic and nonteratogenic lupins . III. Identification of anagyrine as the probable teratogen by feeding trials. J. Tox. Environ. Health . 1, 887-898. Kt~.a, R. F. and Harts, L. D. (1978) Teratogenic effects in cattle of Corium maculatum and corium alkaloids and analogs. Clin . Toxicol. 12, 49-64. Kgt,ea, R. F. (1979) Congenital defects in calves from maternal ingestion of Nicotiana glauca of high anabasine content. Clin . Toxicol. 15, 1726. K1Et.i?tt, R. F., Jags, L. F., Sttue> , J. L. and Vax KAMÍEIV, K. R. (1977) Lupine-induced crooked calf disease and management methods to reduce incidence. J. Range Manage. 30, 97-102 . Km.ea, R. F. and ParrrER, K. E. (1989) Piperidine alkaloid consumption and relation to crooked calf diseaseinducing potential of Lupines formosus . Teratology 40, 42332. Kgt.ex, R. F. and Caowr:, M. W. (1983) Nicotiana glauca-induced swine deformities. Clin. Toxicol. 20, 47-58. Kgt~rt, R. F., Cxowt:, M . W. and LaamEttr, E. A. (1984) Teratogenicity in swine of the Tobacco alkaloid anabasine isolated from Nicotiana glauca . Teratology 30, 61-69. Kgr-za, R. F., Bay-ts, L. D., SrturE, J. L. and CxowE, M. W. (1980) Teratogenicity and toxicity of conüne in cows, ewes and mares. Cornell Vet. 70, 19-26. ParrtEa, K. E., K~t.ea, R. F. and Buctc, W. B. (1985a) Induction of cleft palate in newborn pigs by maternal ingestion of poison hemlock (Corium maculatum) . Am . J. Vet. Res. 46, 1368-1371 . Parrrm, K. E., KEet.ex, R. F. and Buctc, W. B. (1985b) Congenital skeletal malformations induced by maternal ingestion of Corium maculatum (poison hemlock) in newborn pigs. Am. J. Vet. Res. 46, 2064-2066. PaxrEa, K. E., Buxcx, T. D. and Ki:et.ea, R. F. (1988a) Maternal and fetal toxicity of poison-hemlock (Corium maculatum) in sheep. Am . J. Vet. Res. 49, 281-283.
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PertrEte, K. E., Huxcx, T. D., K~e, R. F. and S~ssox, D. V. (198ßb) Radio ultrasound observations of the fetotoxic effects in sheep from ingestion of Corium maculatwn (poison-hemlock) . Clin . Toxicol. 26, 17187. P~Nrlzt, K. E., K~.Ex, R. F. and B~~, D. C. (198ßc) Toxicoses in livestock from the Hemlocks (Corium and Cicuta spp) . J. Arim . Sci. 66, 2407-2413. P~x~rea, K. E., Burtct~t, T. D., Kt=,et.ea, R. F. and Stssox, D. V. (1990) Multiple congenital contractures (MCC) and cleft palate induced in goats by ingestion of piperidine alkaloid~ontaining plants . Reduction in fetal movement as the probable cause. Clic . Tox. 28, 69-84. St~rexn, T. H. (1976) Catalog ojTeratogeric Agents, XI-XVII. Baltimore: Johns Hopkins University Press. SFtve~, J. L., Jeers, L. F, and Btxxs, W. (1967a) Observations on crooked calf disease. JAVMA 151, 91-197 . SHUPE, J. L., Bnvtvs, W., Jeers, L. F. and K~r~x, R. F. (1967b) Lupine, a cause of crooked calf disease. JAVMA 151, 198-203. Sfnrpe, J. L., Jesn~s, L. F., Bnvxs, W. and Kt~, R. F. (1968) Cleft palate in cattle . Cleft Palate J. 1, 346-355. SWINYARD, C. A. and Bcecs:, E. E. (1985) The etiology of arthrogryposes (multiple congenital contracture) . Clic. Orthop. 194, 1 X29. Wusox, J. G. (1973) Normal developmental and susceptible periods. In: Environment and Birth Defects, pp. 121136. New York : Academic Press.
ooai-0im~o s3.ao + .ao
Printed in Cmat Britain .
Pergamoo Prcn p~
REVIEW ARTICLE CONGENITAL SKELETAL MALFORMATIONS AND CLEFT PALATE INDUCED IN GOATS BY INGESTION OF LUPINUS, CONIUM AND NICOTIANA SPECIES K . E . PAIVTER,'
R.
F . KEELER,' T . D .
Bulvcx2 and R. J.
CALLANZ
'USDA/ARS/Poisonous Plant Research Laboratory, 1150 East 1400 North, Logen, UT 84321, and Animal Dairy and Veterinary Science Department, Utah State University, Logen, UT 84322, U.S .A. (Receivedjorpublication 11 1Nay 1990)
R. F . KEELER, T . D . Burrcx and R. J. CALLAN . Review article - Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupines, Corium and Nicotiana species . Toxicon 28, 1377-1385, 1990 .-Three piperidine alkaloid containing plants, Coniem maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupines formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities . Clinical signs of toxicity included those reported in sheep, cattlé and pigs-ataxia, incoordination, muscular weakness, prostration and death . One quinolizidine alkaloid containing plant, Lepinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring. K . E. PANT>~,
INTRODUCTION
Corium macelattan (poison-hemlock), Nicotiana glauca (tree tobacco), Lupines caudatus (tailcup lupine) and Lupines formosus (lunara lupine) have been used to experimentally induce crooked calf disease in cattle (KEELEIt, 1974; KEELER and BALLS, 1978; KEELER, 1979; SHLJPE et al., 1967a,b ; ICEELint et al., 1977; KEEL.E R and PANTER, 1989) . Lupine has also been associated with cleft palate in cattle (ICE~.ER and PANTER, 1989; SxvPE et al., 1968). In addition, Coniem and Nicotiana have induced multiple congenital contracture type skeletal malformations and cleft palate in pigs (KEELER and CROWS, 1983; IC>~LER et al., 1984; PANTER et al., 19ß5a,6) . Research has shown that feeding fresh Corium maculatum to pregnant sheep during 300 days of the gestation period induced transient multiple congenital contractures (MCC) which resolved spontaneously (PANTER et al., 198ßa). They subsequently determined that ConitWn reduced fetal movement in utero (PANTER et al., 19ß8b). Reduction of fetal movement may be a general physiological mechanism for induced MCC in animals and humans according to some workers (SWINYARD and BLECR, 1985; HALL, 1985; BANI{ER, 1985; HAGEetAN and WILLEMSE, 1987) . 1377
137 8
K . E . PANTER
~CH,-CH=-CH, CoIIlhs
Asabaslso
et at.
~CH,-CH,-CH, Y-C~~IeN~o
A~slodsdrloo
FIG . l . FOUR PII~ERIDINE ALKALOIDS PRESENT IN POISONOUS PLANTS KNOWN TO CAUSE CLEFT PALATE Arro MCC rrPE sKELErAL DEPEGTS .
Conüne and y-coniceine arc principal alkaloids in Conium . anabasine in Nicotiana gtauca and ammodendrine in Lapinas jormosus .
Many alkaloids in plants have been implicated as teratogenic agents . Conium maculacontains eight known simple piperidine alkaloids, two of which (conüne and gamma coniceine, Fig. 1) appear to be teratogenic in cattle, pigs and sheep (KEELER, 1974; ICEELER et al., 1980 ; PANTER, Ph.D . Thesis, University of Illinois, 1983) . In Nicotiana glauca the teratogenic agent is a pyridino-piperidine alkaloid, anabasine (Fig. 1) (KEELER, 1979 ; KEELER et al., 1984) . This alkaloid is also present in other plants, including Nicotiana tabacum (burley tobacco) (KEELER et al., 1984) . In lupine, the quinolizidine alkaloid anagyrine is apparently responsible for inducing deformities in cattle (ICEELER, 1976) . KEELER and PANTER (1989) demonstrated that another lupine, Lupines formosus, was teratogenic in cattle and contained piperidine alkaloids whose structures were similar to those in Conium and Nicotiana. The principal alkaloid in Lupines formosus, ammodendrine (Fig. 1) (Frrcx er al., 1974), is believed to be the principle teratogen (ICEELER and tum
PANTER, 1989) . KEEi.ER and BALLS (1978)
orally administered piperidine analogs of conüne to cattle . Results suggested these compounds must possess certain structural characteristics to be teratogenic; e.g. an alpha-substituted piperidine ring that must be saturated or perhaps contain a single double bond, and the alpha side chain must contain at least three carbon atoms. Many of the piperidine alkaloids now known or believed to be teratogenic meet these structural requirements . Livestock vary in their susceptibility to Conium, Nicotiana and Lupines teratogens. KEELER and BALLS (1978) and PANTER et al. (1988c) have shown that the piperidine alkaloids in Conium are most toxic to cattle, and less so to sheep, horses and pigs in that order and are most teratogenic to cattle, and less so to pigs and sheep in that order. Sheep are somewhat resistant to the toxic and teratogenic effects of Conium, Nicotiana and Lupines. Inherent metabolic and pharmacokinetic differences no doubt influence susceptibility among livestock species. An understanding of the variability may help elucidate the nature of poisoning and determine whether metabolism can be altered to reduce incidence, and identify the mechanism of teratogenicity .
Poisonous Plant Induced Malformations
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Teratogenic effects in susceptible animal species are similar and include MCC resulting in fixed extension or flexure of the carpal, tarsal or fetlock joints, enlargement of these joints, lateral deviation of the forelimbs, scoliosis, torticollis, lordosis and cleft palate . We report here experiments to determine the relative toxicity and teratogenicity in goats of Conium maculatum, Nicotiana glauca, Lupimis josmosus and Lupinus caudatus . The usefulness of goats as an alternate animal model for the study of mechanism of action of plant teratogens is considered . MATERIALS AND METHODS The estrous cycles in Spanish-type female goats weighing between 27 kg and 51 kg were synchronized using intravaginal peasaries' containing 45 mg of fluorogestone acetate. Twenty-four to 48 hours after sponge removal all female goats were bred by bucks of e simiL~r type. The female goats were randomly divided into the following five groups : Group 1, six goats, throe gavaged with Coniwn maculation seed (group la) collected in the fall of 1981 in Logan, UT, and stored at 4°C, and three gavaged with fresh Conium maculation (fall regrowth) (group 1b) collected fresh in the same location . In Group 2, 10 goats were gavaged with Nicotimra glauca collected in 1983 near Wickiup, AZ . Group 3 wnsistod of four goats gavaged with Lupinusjormosiu collected at Rio Vista, CA . Group 4 consisted of seven goats gavaged with Luplnur caudaticr collected in 1982 on the Session Mountains east of Woodrufl; UT. Group 5 (wntrol) consisted of nine goats gavaged with water only. All treated goats (Groups 1~) were gavaged with a known amount of plant material suspended in 1-31 of water twice daily for 30 days (30-60 days of gestation). Dosages (Table 1) were selected to assure clinical signs of toxicity in the dams, or were the maximum volume that could be successfully gavagecí . All goats had ad libitum accevs to water, trace mineral salt and alfalfa hay. Clinical signs of toxicity were observed and rewrded . At parturition, teratogenic effects in offspring wen assessed by two scientists, one of whom was unaware of treatment regimes in order to eliminate bias. Photographs and X-rays wen taken of severely deformed fetuses (Figs 2~) . RESULTS
Group 1--Conium maculatuur Clinical signs of maternal toxicity were modest to severe in goats gavaged with seed or fresh Conium maculatum, depending on the dosage . The most severe clinical signs occurred about 15 min to 2 hr after treatment and muscular weakness appeared to persist until the next dosage period . Signs of toxicity included trembling, muscular weakness and fasciculations, depression, ataxia and collapse. Clinical signs of toxicity were the same for seed and fresh plant. After the initial phase of the toxicity (about 2 hr) the goats' appetites were good and they often ate in a sternal recumbent position when hay was offered. They readily moved about but could stand for only X10 min early in the daily expression of toxicoses but could stand longer after several hours. More plant material was required to maintain the same clinical effect as the days passed during the experiment, suggesting ruminal or hepatic adaptation might be occurring. All clinical signs of toxicity in dams disappeared 248 hr after final treatment. The single goat fed Conium seed had triplets ; all had bilateral cleft palate (Fig. 2) and severe MCC (Figs 3,4). All were dead at birth. All kids had variable degrees of the following malformation expressions: fixed carpal flexure and rotation, elbow and shoulder joint rigidity, fixed and rigid stifle joints, severely flexed hock joints, rib cage deformities, sternal deviation, overextended fetlocks in the forelimbs, lateral rotation or rigidity of 'Pre-prepared pessaries were purchased from Intervet, B. P. 235-45 Avenue Joxi, 49002 Angers Cedex, France. Mention of a proprietary product does not wnstitute a guarantce or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
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F~a . 2 . Bilateral cleft palate induced by Conium aced, N. glauca and L . jormosus .
hock, stifle, and pastern joints in the rear limbs, and deviated spinal column with scoliosis, torticollis or lordosis . One of two goats gavaged with fall growth Conium had twins with variations of modest carpal flexure and elbow joint rigidity that were otherwise normal. The other goat had twins, one of which had equivocal carpal flexure and the other had modest carpal flexure and modest elbow rigidity, but appeared to be normal otherwise. Seed and fresh plant induced the same signs of toxicity, but signs persisted longer in the goat gavaged with seed . The persistence of the clinical signs appeared to parallel the severity of the malformations. Group 2-Nicotiana glauca Clinical signs of toxicity in ten goats gavaged Nicotiana glauca were moderate to severe and persisted between doses of plant material. The first signs of toxicity were evident about 15 min after treatment and were most severe by 1-2 hr after treatment. Signs of toxicity included muscular weakness in the legs and neck, ataxia, incoordination, muscular fasciculations, collapse and sternal recumbency. All but one of the 16 kids born to these goats had bilateral cleft palates and varying degrees of MCC type skeletal malformations. The malformations were the same as those described for Conium seed and are illustrated in Figs 2~. The severity of the malformations paralleled the severity of the clinical signs of toxicity in the dam. Group 3-Lupinus formosus Clinical signs of toxicity were slight to severe in four goats gavaged Lupinus formosus and were similar to those described for Conium maculatum and Nicotiana glauca, however, because of plant volume we were unable to increase dosages in some of the goats to
Poisonous Plant Induced Malformations
FIG. 3. OVEIiEICIENSION AT THE PASTIItN JOINTS, SEVERE FD® CARPAL FLEXURE, RIH CAGE DEPRESSION AND SPINAL COLUI~i DEPRL~ON (LORDOSiS) (ARROWS) TYPICAL IN RIDS FED Conitan SEED, N . giQüCO AND L . forIltOS7L4 .
Severity and location of malformation varied from kid to kid.
FIG . 4. PHOTOGRAPH of AN X-RAY OF A Rm IN A VENTRAL DORSAL VQ;W WITH THE HEAD EXTENDED .
Note the deviation and the severe curvature of the thoracic region, the wedging of the vertebrae and the abnormal rib and rib ar8e development .
138 1
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TABLe 1 . TOXICITY AND TIIüTOGEPIIC RE4IJL1'3 OF I~DING Conium maculatum, Nicotiana AND Lupinus CauL%atuS TO PItFX3NANT GOATS
No. of goats (mean wt, kg)
I (37) 2 (36) 10 (36) 4 (43) 7 (40) 9 (42)
Treatment
C. maculatuur (seed) C. maculatuur (fresh) N. glauca L. jormosus L. caudatus Control
Clinical toxicoses
Moderate to severe Moderate to severe Moderate to severe Slight to severe None to slight -
'Mean daily dosage g
130 387 200 530 500 -
glauca, Lapinasjormosas
Teratogenic results tNo . CP
$No. MCC
3/3 0/4 15/16 §3/9 0/14 0/20
3/3 3/4 15/16 4/9 0/14 0/20
'Daily dosage was the total of the a .m . and p .m. dosages . The dosage of fresh plant was adjusted daily due to variability in toxicity of material . tCP = cleft palate, all were bilateral cleft except 1 kid in the L . jormosus which was a unilateral cleft . $MCC = multiple congenital contractures and indicates the number of kids with MCC per number of kids born. §One goat died on day 36 of gestation, she had triplets but palate or skeletal defects were not discernible because of age and size of the fetuses . We would not expect closed palates until after 38 days gestation .
maximize clinical signs of toxicity (Table 1). Two of four goats had deformed offspring . Two kids from the same dam had bilateral cleft palates. One of two kids from another dam had a unilateral cleft palate, but the palate of its twin was normal . The skeletal defects were severe and similar to those described for Conium and N. glauca in the two kids with bilateral cleft palate . Both twins, one of which had the unilateral cleft palate, had moderate carpal flexure, but no other skeletal anomalies. The severity of the clinical toxicoses in dams paralleled the severity of the subsequent malformations in the fetuses. Group 4-Lupinus caudatus
Only slight clinical signs of toxicity appeared in two of seven pregnant goats gavaged
Lupinus caudatus. They exhibited mild muscular weakness in the cervical region and
carried their heads lower than other goats exhibiting the early stages of toxicity . These signs were evident only during the first few days of treatment, and did not persist between doses. These seven goats had 14 kids and all were normal with no skeletal or palate defects. Group S~ontrols
All nine control goats had normal kids (20 total) without cleft palate or skeletal abnormalities. DISCUSSION
Conium maculatum, Nicotiana glauca and Lupinus jormosus, all of which contain piperidine alkaloids, induced congenital birth defects in pregnant goats when gavaged during gestation days 30-60. The birth defects included cleft palate and MCC that were expressed directly or indirectly as flexure of the joints, torticollis, scoliosis, lordosis, rib cage depressions and wedging of vertebrae. The quinolizidine alkaloid-containing plant
Poisonous Plant Induced Malformations
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Lupines caudatus, however, did not induce any malformation in offspring when goats were gavaged during the same time period . The results showed that Conium maculatum, Nicotiana glauca, and Lupines formosus induce cleft palate and MCC in goats similar to deformities reported in cows and pigs suggesting that goats may be a useful alternate animal model to study plant teratogens and mechanisms of fetal effects. Our results presented here and elsewhere (KEELER and Pnrr~rm, 1989; Pnrr~t~It et al., 1990) suggest that livestock species either differ in metabolism of or in absorption and transfer of these plant toxins to the fetus. The three plants containing piperidine alkaloids, Conium, Nicotiana, and Lupinesformosus, induced cleft palate and MCC in the goats, but Lupines caudatus, which contains quinolizidine alkaloids did not cause significant maternal toxicity or teratogenicity in the goat. These and other findings suggest that piperidine alkaloids may be teratogenic in both species (cow and goat) but that the quinolizidine alkaloid anagyrine is teratogenic only in the cow. This species difference is interesting and suggests possible ruminal or liver metabolic processing differences. ICEELER and BnLIs (1978) proposed that certain minimum structural characteristics of piperidine alkaloids were required for teratogenicity in cattle . The structural requirements included an alphasubstituted piperidine ring whose alpha side chain was at least three carbons long. IC>~r m and Pntv~It (1989) hypothesized that, in the cow, minor metabolic changes in the quinolizidine alkaloid anagyrine from lupine could result in a piperidine alkaloid that meets the structural requirements for teratogenic piperidines. Thus, a piperidine alkaloid meeting these structural requirements may be the proximal teratogenic compound even when the quinolizidine, anagyrine is the suspected agent in the plant. This might explain why terata are absent in goats fed the anagyrine containing Lupines caudatus even though the plant causes cleft palate and MCC in cattle . Goats may not metabolically modify anagyrine in the rumen to a teratogenic piperidine as cows hypothetically do. Further research is needed to elucidate this possibility. The present results showed that MCC may occur alone or with cleft palate depending on the stage of pregnancy when exposure occurs . In animals and humans, the biological mechanisms) causing cleft palate and MCC type skeletal abnormalities are not fully elucidated. Cleft palate induction may involve the same biological mechanism as MCC induction even though the insult occurs at a different gestational period (see below) . MCC and cleft palate were here associated with plant toxins administered at different gestational periods. Pnrr~rEtt et al. (1985a) showed that only cleft palate occurred when swine were fed Conium maculatum during days 305 of gestation, and that feeding this plant during days 43-53 of gestation did not cause cleft palate but did cause MCC type limb and spinal malformations and associated rib cage abnormalities (PAIVTER et al., 19856) . When the same plant was fed from days 511 of gestation, the limb defects were less severe and rear limbs were effected more often then the forelimbs. As expected, (WILSbN, 1973; Sl~nxn, 1976) stage of gestation when exposure occurred determined terata expression. A correlation between a reduction in fetal movement and associated MCC and cleft palate by these plants and the alkaloids therefrom has been demonstrated (PAIVT'ER et al., 1988b; PANTER et al., 1990). In 1988, Pnrr~It et al. reported that Conium maculatum inhibited fetal movement in sheep for about 5-6 hr after each dose, before returning to normal by 12-16 hr. Fetuses born to ewes so dosed for several days showed modest carpal flexure, which resolved spontaneously by 8 weeks, and no cleft palate . We believed that the lack of severe birth defects (MCC and cleft palate) in those sheep resulted from intermittent rather than constant inhibition of fetal movement during the critical gestational period. Subsequently, PnN~It et al. (1990), and as reported here, showed that prolonged
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inhibition of fetal movement induced by Corium maculattim and Nicotiana glauca during critical stages of gestation resulted in cleft palate and MCC. Thus, duration and severity of fetal movement inhibition are directly correlated with deformity expression severity suggesting a mechanistic association. The relative roles of the primary pathologic condition or biochemical lesion and loss of fetal movement in the expression of MCC and cleft palate are not known. Further research is needed to clarify the teratogenic role of chemicals which could affect fetal movement at a critical stage of development but not cause primary pathologic changes. The goat is a good animal model for monitoring fetal movement with ultrasound because of size similarity to humans and because goats are docile. The association between loss of fetal movement and plant-induced birth defects must be clarified to be sure that the loss of movement is not independent of malformations and that these malformations do not occur with normal fetal movement . We suggest in the present experiment that the MCC resulted from loss of fetal movement induced by the CNS depressing effect of these plant alkaloids. We also suggest that the cleft palate was a direct result of mechanical obstruction of the tongue resulting from the same CNS depressing effect and lack of fetal activity . Acknow/edgements~ontribution of the USDA/ARS Poisonous Plant Research Laboratory in cooperation with the Utah Agricultural Experiment Station, Utah State University, Logan, UT 84322. Approved as Journal paper no . 3822 . The authors wish to thank LBw Det.[. Bat.ts, Tort Buctc, A~ Mac~ut,ts and Lt7w KEtso for animal care and assistance and Ms Tt~ WIERENGA for technical assistance . REFERENCES Barrxex, B. Q. (1985) Neuropathologic aspects of arthrogryposis multiplex congenital . Clip. Orthop . 194, 303. Frrctt, W. L., DOLINGI~t, P. M. and Drnn~eci, C. (1974) Alkaloid studies. LXVIII . Novel piperidyl alkaloids from Lupinesformosus. J. Org. Chem . 39, 2974-2979. HAGEMAN, G. and Wtt i a..aF, J. (1987) The pathogenesis of fetal hypokinesia. A neurological study of 75 cases of congenital contractur~es with emphasis on cerebral lesions. Neuropediarrics 18, 22-33 . HALL, J. G. (1985) Genetic aspects of arthrogryposis . Clip . Orthop . 194, 44-53. Kt~t~x, R. F. (1974) Conüne, teratogenic principal from Corium maculatum producing congenital malformations in calves. Clip. Toxicol. 7, 19206 . KEet .ea, R. F. (1976) Lupin alkaloids from teratogenic and nonteratogenic lupins . III. Identification of anagyrine as the probable teratogen by feeding trials. J. Tox. Environ. Health . 1, 887-898. Kt~.a, R. F. and Harts, L. D. (1978) Teratogenic effects in cattle of Corium maculatum and corium alkaloids and analogs. Clin . Toxicol. 12, 49-64. Kgt,ea, R. F. (1979) Congenital defects in calves from maternal ingestion of Nicotiana glauca of high anabasine content. Clin . Toxicol. 15, 1726. K1Et.i?tt, R. F., Jags, L. F., Sttue> , J. L. and Vax KAMÍEIV, K. R. (1977) Lupine-induced crooked calf disease and management methods to reduce incidence. J. Range Manage. 30, 97-102 . Km.ea, R. F. and ParrrER, K. E. (1989) Piperidine alkaloid consumption and relation to crooked calf diseaseinducing potential of Lupines formosus . Teratology 40, 42332. Kgt.ex, R. F. and Caowr:, M. W. (1983) Nicotiana glauca-induced swine deformities. Clin. Toxicol. 20, 47-58. Kgt~rt, R. F., Cxowt:, M . W. and LaamEttr, E. A. (1984) Teratogenicity in swine of the Tobacco alkaloid anabasine isolated from Nicotiana glauca . Teratology 30, 61-69. Kgr-za, R. F., Bay-ts, L. D., SrturE, J. L. and CxowE, M. W. (1980) Teratogenicity and toxicity of conüne in cows, ewes and mares. Cornell Vet. 70, 19-26. ParrtEa, K. E., K~t.ea, R. F. and Buctc, W. B. (1985a) Induction of cleft palate in newborn pigs by maternal ingestion of poison hemlock (Corium maculatum) . Am . J. Vet. Res. 46, 1368-1371 . Parrrm, K. E., KEet.ex, R. F. and Buctc, W. B. (1985b) Congenital skeletal malformations induced by maternal ingestion of Corium maculatum (poison hemlock) in newborn pigs. Am. J. Vet. Res. 46, 2064-2066. PaxrEa, K. E., Buxcx, T. D. and Ki:et.ea, R. F. (1988a) Maternal and fetal toxicity of poison-hemlock (Corium maculatum) in sheep. Am . J. Vet. Res. 49, 281-283.
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PertrEte, K. E., Huxcx, T. D., K~e, R. F. and S~ssox, D. V. (198ßb) Radio ultrasound observations of the fetotoxic effects in sheep from ingestion of Corium maculatwn (poison-hemlock) . Clin . Toxicol. 26, 17187. P~Nrlzt, K. E., K~.Ex, R. F. and B~~, D. C. (198ßc) Toxicoses in livestock from the Hemlocks (Corium and Cicuta spp) . J. Arim . Sci. 66, 2407-2413. P~x~rea, K. E., Burtct~t, T. D., Kt=,et.ea, R. F. and Stssox, D. V. (1990) Multiple congenital contractures (MCC) and cleft palate induced in goats by ingestion of piperidine alkaloid~ontaining plants . Reduction in fetal movement as the probable cause. Clic . Tox. 28, 69-84. St~rexn, T. H. (1976) Catalog ojTeratogeric Agents, XI-XVII. Baltimore: Johns Hopkins University Press. SFtve~, J. L., Jeers, L. F, and Btxxs, W. (1967a) Observations on crooked calf disease. JAVMA 151, 91-197 . SHUPE, J. L., Bnvtvs, W., Jeers, L. F. and K~r~x, R. F. (1967b) Lupine, a cause of crooked calf disease. JAVMA 151, 198-203. Sfnrpe, J. L., Jesn~s, L. F., Bnvxs, W. and Kt~, R. F. (1968) Cleft palate in cattle . Cleft Palate J. 1, 346-355. SWINYARD, C. A. and Bcecs:, E. E. (1985) The etiology of arthrogryposes (multiple congenital contracture) . Clic. Orthop. 194, 1 X29. Wusox, J. G. (1973) Normal developmental and susceptible periods. In: Environment and Birth Defects, pp. 121136. New York : Academic Press.
