Induction of terata in hamsters by solanidane alkaloids derived from Solanum tuberosum.

The potential induction of terata by solanidanes has been of public health concern since a report in 1972 hypothesized that certain birth defects in humans could be attributed to ingestion of blighted potatoes. The potential teratogenicity of solanidane alkaloids from potatoes and tomatoes in domestic livestock had been considered even earlier. In the present report, oral administration of the steroidal alkaloid glycosides alpha-solanine and alpha-chaconine and their aglycone solanidine is shown to induce craniofacial malformations (exencephaly, encephalocele, and anophthalmia) in Syrian hamsters. All three alkaloids, that were either isolated or obtained by hydrolysis from Solanum tuberosum (var. Kennebec) sprouts, possessed the 22-(R),25(S)-configuration in the indolizidine moiety with no other isomers present. Toxicity constraints precluded administration of dosages high enough to induce statistically significant levels of terata in litters dosed with alpha-chaconine and permitted the attainment of only marginal statistical significance for alpha-solanine. However, malformation induction at p < 0.005 was observed in litters upon dosing both the nontoxic aglycone solanidine and the derivative solanidine N-oxide at higher levels. The relatively high teratogenicity of nontoxic solanidine, compared to the glycosides, demonstrates that terata induction by solanidanes is not due to maternal toxicity nor is the oligosaccharide portion of steroidal alkaloid glycosides required to facilitate passage of the teratogen to the fetus. The teratogenicity of solanidine N-oxide, a putative metabolite, suggests that N-oxidation is not an effective mammalian detoxification pathway. Relative teratogenic potencies (RTP) were assigned to solanidanes by conversion of literature data to equimolar doses compared to the powerful Veratrum teratogen jervine and the nonteratogenic spirosolane tomatidine. RTP values are as follows: jervine (100), 22(S),-25(R)-solanidanes (50), alpha-chaconine (43), alpha-solanine (32), 22(R),25(S)-solanidine (32), solanidine N-oxide (32), 5 alpha,6-dihydrosolanidine (9), and tomatidine (0).

Quinolizidine and piperidine alkaloid teratogens from poisonous plants and their mechanism of action in animals.

Quinolizidine and piperidine alkaloid teratogens from Lupinus, Conium, and Nicotiana genera have been identified as causes of birth defects in livestock induced by poisonous plants. Many defects now known to be related to poisonous plant ingestion were once thought to have a genetic origin. This supposition delayed diagnosis, reporting, and understanding of such birth defects, because breeders and producers feared the news would make it difficult to sell breeding stock. Defects caused by quinolizidine and piperidine teratogens include cleft palate and contracture-type skeletal defects such as arthrogryposis, scoliosis, torticollis, and kyphosis. Teratogens have been identified, differences in susceptibility to teratogenic compounds among livestock species have been elucidated, periods of gestation when specific types of birth defects occur have been determined, and information about mechanism of action has been developed.

Concentration of galegine in Verbesina encelioides and Galega oficinalis and the toxic and pathologic effects induced by the plants.

Verbesina encelioides administered to sheep by gavage induced clinical signs of toxicity and pathologic lesions identical to those induced by Galega officinalis. Sheep had compromised respiratory function with shallow, rapid respiration and frothy exudate from the nares. Affected animals necropsied at time of death presented with hydrothorax with as much as 2 to 3 L of straw-colored thoracic fluid with fibrin tags and congestion and edema of the lungs. The trachea and lung airways contained frothy material with fibrin strands. In some cases, subendocardial hemorrhage of the left ventricle was present. Galegine, a guanidine compound believed to be responsible for these effects, was found at an average concentration of about 0.46% in Galega and at 0.08% in the Verbesina collection that induced toxicosis. While G. officinalis is a known poisonous plant, its very limited distribution in the U.S. causes it to be of minor importance. V. encelioides, however, is widely distributed in the U.S. and presents a potential hazard for grazing livestock. Verbesina may have been responsible for past livestock deaths in the U.S., and thus should be classified as a poisonous plant.

Myopathy in cattle induced by alkaloid extracts from Thermopsis montanta, Laburnum anagyroides and a Lupinus sp.

A purified alkaloid preparation containing N-methylcytisine, cytisine, 5,6-dehydrolupanine, thermopsine and anagyrine from Thermopsis montana induced prolonged recumbency and microscopic acute hyaline skeletal myodegeneration with myofibre regeneration in cattle similar in type and severity to that induced by Thermopsis montanta plant material. This indicates that the alkaloid(s) of Thermopsis montana are responsible for the myopathy caused by the plant. An alkaloid preparation containing mostly anagyrine from a Lupinus sp. and an alkaloid preparation containing only cytisine from Laburnum anagyroides each caused microscopic skeletal muscle degeneration and necrosis similar to, but less severe than, the alkaloid extract from T. montana, but without clinical recumbency. Dosage and severity of response suggest that neither of those two alkaloids alone can account for the effects induced by Thermopsis. The data suggest that quinolizidine alkaloids with a alpha-pyridone A-ring may be responsible for the lesions and that individual alpha-pyridones may have additive effects.

Expression of Veratrum alkaloid teratogenicity in the mouse.

Jervine, a steroidal alkaloid found as a minor constituent in the teratogenic range plant Veratrum californicum, has produced similar terata in sheep, rabbit, hamster, and chick, although the sensitivity to the alkaloid varies in the different species. Sprague Dawley rats and Swiss Webster mice are relatively insensitive. The aim of this study was to determine the teratogenic potential of jervine in three strains of mice and to ascertain if the response is strain dependent. One strain, Swiss N:GP(S), was retested since a Swiss Webster strain had been found previously to be jervine-resistant. In addition, we tested C57BL/6J and A/J, which are known to differ in their response to the teratogenic action of steroids and vitamin A. Mice were treated by gavage with single doses of jervine (70, 150, or 300 mg/kg body weight) on either day 8, 9, or 10 of gestation. Jervine was teratogenic to C57BL/6J and A/J mice but not to N:GP(S). The induced terata included cleft lip with or without cleft palate, isolated cleft palate, mandibular micrognathia or agnathia, and limb malformations. Fetal teratogenicity and maternal and fetal toxicity were highly correlated. The prevalence of each defect and fetal death was a function of strain, dose, and time of treatment. Maternal death was higher in C57BL/6J than in A/J mice. Although some of the terata were similar, the response pattern between strains was different from corticosteroids and vitamin A for both sensitive period and the strain dose response. An effect on differentiation of chondrocyte precursors may account for many of the defects, but an earlier lethal effect on differentiation of neural crest cells or precordal mesenchyme may also occur.

Early embryonic death in lambs induced by Veratrum californicum.

Both field and laboratory observations have suggested the possibility of embryonic death from 14th day ingestion of Veratrum californicum by pregnant sheep. An experiment was conducted to tabulate the incidence of embryonic death from 14th day ingestion of high doses of plant material. Six of eight ewes so dosed were non-pregnant at term (75%), compared to none in controls, and were, therefore, believed to have experienced embryonic death from the plant ingestion. Results suggest that ingestion of plant material could account for many open ewes at lambing time in ranching operations where Veratrum has been ingested in early gestation.

Multiple congenital contractures (MCC) and cleft palate induced in goats by ingestion of piperidine alkaloid-containing plants: reduction in fetal movement as the probable cause.

Fetal movement, observed by ultrasound imaging, was significantly reduced (P less than or equal to 0.001) in pregnant goats gavaged with Conium seed and Nicotiana glauca and temporarily reduced with fresh Conium plant. Conium seed and Nicotiana glauca induced cleft palate and multiple congenital contractures in 100% of the kids born to pregnant goats gavaged with these plants. Multiple congenital contractures included torticollis, scoliosis, lordosis, arthrogryposis, rib cage anomalies, over extension, and flexure and rigidity of the joints. However, in goats gavaged with fresh Conium plant, fetal movement was inhibited for only about 5 hours after each individual dosage and gradually returned to control levels 12 hours after dosing. Fetal malformations in this group were limited from modest to moderate contractures of the front limbs, which resolved by 8-10 weeks post partum. No cleft palates were induced. Fetal movement was not inhibited in goats fed Lupinus caudatus and no cleft palates or multiple congenital contractures were induced in their offspring. The duration of the reduction in fetal movement appears to be an important factor in the severity and permanence of the deformities, particularly with cleft palate, spinal column defects, and severe joint deviation and fixation.

Spirosolane-containing Solanum species and induction of congenital craniofacial malformations.

Comparison by GC analysis of purified alkaloid extracts of Solanum species revealed no measurable free solasodine, other spirosolanes, or any non-spirosolane steroidal alkaloid aglycones in unhydrolyzed total alkaloid fractions of fruit of Solanum elaeagnifolium Cav. (silverleaf nightshade), Solanum sarrachoides (S. villosum Lam.--hairy nightshade), Solanum dulcamara L. (European bittersweet nightshade) or Solanum melongena L. (eggplant). All alkaloidal material was apparently present as glycoside. Conversely, sprouts of Solanum tuberosum L. (potato) contained 67% of its alkaloids as glycosides, which was freed only upon hydrolysis with the remaining 33% present as free solanidine. GC/MS analysis of hydrolysates of purified extracts of the test Solanum species revealed that solasodine was a principal or sole aglycone of the alkaloid glycosides in each of the test species except Solanum tuberosum. In the latter, solanidine was the sole aglycone. Among the test species, exclusive of S. tuberosum, only S. dulcamara contained aglycones other than solasodine. In addition to solasodine, S. dulcamara contained appreciable amounts of an unknown spirosolane, an aglycone provisionally identified as soladulcidine. The induction of congenital craniofacial malformations in hamsters by high oral doses of the four Solanum species that contained mainly solasodine glycosides--S. elaeagnifolium, S. dulcamara, S. sarrachoides and S. melongena was compared to inductions of malformations by Solanum tuberosum, that contained mainly solanidane glycosides. Compared to controls, Solanum elaeagnifolium and Solanum dulcamara fruit both induced a high percentage incidence of deformed litters (20.4 and 16.3, respectively) that was statistically significant (P less than 0.001 level) while percentage incidence of deformed litters induced by Solanum sarrachoides and Solanum melongena fruit (9.5 and 7.6 respectively) were both higher than controls (3.4%), in neither case was the incidence statistically significant (P less than .05). Deformed litter incidence induced by sprouts of Solanum tuberosum was 24.0%, (P less than 0.001).

Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus, Conium and Nicotiana species.

Three piperidine alkaloid containing plants, Conium maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupinus 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, cattle and pigs--ataxia, incoordination, muscular weakness, prostration and death. One quinolizidine alkaloid containing plant, Lupinus 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.

Oral, osmotic minipump, and intramuscular administration to sheep of the Veratrum alkaloid cyclopamine.

Logistic and biologic aspects of three separate means of administration of cyclopamine for experimental induction of terata or embryonic death in sheep were examined. Oral capsule administration of crystalline cyclopamine is logistically simple and biologically effective, but costly in terms of amount of compound required. Embryos were affected in five of seven ewes dosed cyclopamine orally at higher levels (four nonpregnant and one with cyclopia). Intramuscular administration of cyclopamine dissolved in ethanol was logistically simple but without biologic effect at levels tested. Three of three treated ewes had normal offspring. Osmotic minipump administration of powdered cyclopamine suspended in propylene glycol was logistically unsatisfactory with serious delivery complications. Osmotic minipump administration of cyclopamine dissolved in ethanol was logistically very satisfactory, and one ewe among three treated animals was nonpregnant at term. There were no nonpregnant ewes nor deformed offspring in 17 controls.

Piperidine alkaloid composition and relation to crooked calf disease-inducing potential of Lupinus formosus.

A congenital deformity condition called crooked calf disease, of widespread occurrence in western North America, is known to be induced by maternal ingestion during gestation of certain members of the Lupinus genus containing the quinolizidine alkaloid teratogen anagyrine. Because some piperidine alkaloids from other sources induce a similar condition, we have investigated the alkaloid composition and teratogenicity of Lupinus formosus, reported by others to be low in quinolizidines but rich in the type of piperidine alkaloids that we have speculated would be teratogenic. GC/MS analysis of L. formosus showed seven major and nine minor components in the total alkaloid fraction. All seven major and five of the nine minor components, representing all but 3% of the fraction, were identified by mass spectrometric fragmentation patterns and GC retention times. They included several potentially teratogenic piperidine alkaloids (including a very large amount of ammodendrine), as well as several nonteratogenic quinolizidine alkaloids plus a trace (at nonteratogenic levels) of the known quinolizidine teratogen anagyrine. The plant induced severe crooked calf disease with limb, spinal, and palate involvement in experimental calves. The deformities are believed to have been induced by ammodendrine.

Thermopsis montana-induced myopathy in calves.

Six heifer calves were administered the dried and ground whole plant of Thermopsis montana by gavage once daily for 2 to 4 days at an initial dosage rate of 1 g/kg of body weight. Two of the 6 heifers died after the second dose, and the remaining 4 calves were humanely killed and their tissues were examined. Serum creatine kinase and aspartate transaminase activities were significantly (P less than 0.05) increased after initiation of plant administration. All calves had skeletal muscular degeneration or evidence of regeneration and repair of damaged myofibers in all skeletal muscle groups examined. Myoglobinuria and cardiac muscle degeneration were not detected in any of the calves.

Pathology in hamsters administered Solanum plant species that contain steroidal alkaloids.

Syrian hamsters were orally administered ground plant material from either Solanum sarrachoides, S. melongena, S. eleagnifolium, or S. dulcamara. Six of eight hamsters administered S. eleagnifolium and eight of 10 hamsters administered S. dulcamara died following administration of plant material and had gastric glandular mucosal necrosis and small intestinal mucosal necrosis with little inflammation. Hamsters administered S. sarrachoides or S. melongena did not die and had only lesions compatible with gastric distension. Both S. eleagnifolium and S. dulcamara contained solasodine glycoalkaloids(s), and S. dulcamara also contained an equal amount of other glycoalkaloids which were probably derived from soladulcidine (dihydrosolasodine). The lesions produced by these two plants were similar to those reported earlier to be caused by sprout material of S. tuberosum (in which solanidane alkaloids predominate) and by an alkaloid extract of S. tuberosum sprouts. Because of similarities in saponin-like activity and structure of solasodine glycoalkaloids to the solanidine glycoalkaloids of potato sprouts, the glycoalkaloids of S. dulcamara and S. eleagnifolium were probably the agents responsible for the lesions observed.

Individual animal susceptibility and its relationship to induced adaptation or tolerance in sheep to Galega officinalis L.

Oral administration of Galega officinalis L to sheep demonstrated a marked variation in individual animal susceptibility to the toxic effects of the plant. As little as 5 g/kg of dried ground plant induced moderate tracheal frothing in 1 ewe while nearly 5 times that amount failed to elicit any recognizable toxic effects such as frothing, pulmonary edema or hydrothorax in others. Ten g/kg induced severe effects in 3 ewes. Ewes administered levels of plant between 5 and 24 g/kg had toxic effects whose severity was often unrelated to level administered. There was no apparent difference in average severity of clinical signs of toxicity nor pathologic lesions to challenge doses of 24 g/kg of the plant between groups of ewes with an immediate previous history of increasing doses of the plant and others with no history of ingesting the plant. Previously reported apparent induced adaptation or tolerance to G officinalis L in some animals is more likely to have been a result of the extreme variation in individual animals susceptibility.

Toxicoses in livestock from the hemlocks (Conium and Cicuta spp.).

The hemlocks, Conium maculatum (poison-hemlock) and Cicuta spp. (waterhemlock), are poisonous plants that cause sizeable losss to the livestock industry. Clinical signs of poisonhemlock toxicosis are similar in all species of livestock and include muscular weakness, incordination, trembling, initial central nervous system stimulation, depression and death from respiratory paralysis. Poison-hemlock also causes skeletal defects in the offspring of cattle, pigs and sheep and cleft palate in pigs when ingested during specific periods of gestation. The primary toxicants in poison-hemlock are coniine and gamma-coniceine. Coniine predominates in mature plants and seed, whereas gamma-coniceine predominates in early growth of the plant. Waterhemlock is the most violently toxic poisonous plant known. The toxicant is cicutoxin, which acts on the central nervous system, causing violent convulsions and death. Clinical signs of poisoning appear within 15 min after ingestion of a lethal dose and include excessive salivation, nervousness, tremors, muscular weakness and convulsive seizures interspersed by intermittent periods of relaxation and a final paralytic seizure resulting in anoxia and death. Elevated activities of lactic dehydrogenase, aspartate aminotransferase and creatine kinase in blood are observed, indicative of muscular damage. Toxicoses from poisonhemlock and waterhemlock generally occur in early spring when both plants emerge before other, more palatable plants begin to grow. All parts of the poison-hemlock plant are toxic. The root or tubers of waterhemlock are toxic; however, experimental evidence concerning the toxicity of other plant parts is inconclusive.

Livestock models of human birth defects, reviewed in relation to poisonous plants.

Certain birth defects in livestock induced by poisonous plants possess clinical similarities to congenital deformities in humans. They meet some criteria as models for those corresponding human conditions. They are induced by several plants, including members of the Astragalus, Lupinus, Conium, Nicotiana and Veratrum genera. The terata expressions include effects in bone and soft tissue, particularly in the limbs, spinal cord and cephalic regions. Whether these livestock conditions become extensively used as models of human terata will be determined by how well they meet the criteria sought in good models. These prospective models have many favorable characteristics; however, being large domestic livestock, they have certain logistic disadvantages compared with small rodents as models.

Maternal and fetal toxicity of poison hemlock (Conium maculatum) in sheep.

Poison hemlock (Conium maculatum) was toxic to pregnant ewes and their fetuses when fed during gestation days 30 through 60. Maternal effects included trembling, muscular weakness in the neck initially, then progressing to the limbs, ataxia, frequent urination and defecation, and death. Convulsive seizures were not observed. Fetotoxic effects included excessive flexure of the carpal joints with lateral deviation in the front limbs and kinked tails. At term, 7 of 11 lambs had varying degrees of the limb abnormalities, but all lambs appeared clinically normal at 8 weeks after parturition.

Mechanism of death in Syrian hamsters gavaged potato sprout material.

Greened or sprouted potatoes contain increased concentrations of steroidal alkaloids that have caused intoxication and death in a wide variety of animal species, however, the cause of death in these animals has not been determined. Potato alkaloids can cause death when parenterally administered, and is attributed to the acetylcholinesterase inhibitory activity of solanine and chaconine. To determine the cause of death in animals ingesting potato sprout material, 40 Syrian hamsters were divided into 4 equal groups and gavaged once on day 0 either water, 300 mg of potato sprout material, 400 mg of potato sprout material, or 500 mg of potato sprout material. Tissues were examined grossly and microscopically at 72 hr post-gavaging and brain acetylcholinesterase activity of each hamster was measured. The 300-mg dose group had increased mean acetylcholinesterase activity compared with control hamster mean activity, and the 400-mg and 500-mg dose groups had 90% and 84% of the mean acetylcholinesterase activity of the control hamster mean activity. There was severe gastric and proximal small intestinal mucosal necrosis in those hamsters which died prior to euthanasia. Additionally, several hamsters had valvular endocarditis and infarcts. Death could not be attributed to the slight acetylcholinesterase inhibition in the 2 higher dose hamster groups and was related to the severe gastrointestinal necrosis which occurred in hamsters of these groups.