Submitted to: Journal of Applied Toxicology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2009
Publication Date: 3/16/2009
Citation: Welch, K.D., Panter, K.E., Lee, S.T., Gardner, D.R., Stegelmeier, B.L., Cook, D. 2009. Cyclopamine-induced Synophthalmia in Sheep: Defining a Critical Window and Toxicokinetic Evaluation. Journal of Applied Toxicology, 29:414-421. (www.interscience.wiley.com) DOI 10.1002/jat.1427
Interpretive Summary: Early evaluation of the chronology of teratogenicity of V. californicum in sheep indicated that the plant must be ingested between gestation days (GD) 10-15 in order to cause synophthalmia malformations. It was later reported that GD 14 is the critical day for synophthalmia malformations to occur, as it was observed that ewes dosed with V. californicum on GD 11-13 and 15-16 all had normally developed fetuses. These data suggest that the critical window for synophthalmia formation is approximately one day. The objective of this study was to better define the critical window of susceptibility and relate this susceptibility to the toxicokinetics of the Veratrum alkaloid, cyclopamine. The results from this study support previous findings that the critical window for synophthalmia formation is short. Even though we found that ewes dosed on GD 13 in addition to GD 14 are susceptible to synophthalmia formation, the exact time of conception is still inaccurate and consequently with the methods employed here we cannot more accurately define the critical window. The pharmacokinetic analysis demonstrated that the elimination of cyclopamine from sheep is very quick indicating that the plant must be consumed in sufficient quantities during the very narrow critical window for teratogenesis to occur. Also, the observations made in this study suggest that severely deformed lambs develop at a much slower rate than less deformed lambs and that deficiencies in placentation may play a role.
Technical Abstract: BACKGROUND: Cyclopamine, a steroidal alkaloid, from the plant Veratrum californicum is teratogenic in sheep causing a series of craniofacial, skeletal, and other defects. The critical window for cyclopamine-induced synophthalmia formation was reported in the 1950s to be gestational day (GD) 14. The purpose of this work is to better describe the extent of cyclopamine-induced teratogenesis in sheep, to better define the window of susceptibility to synophthalmia formation, and to characterize the pharmacokinetics of cyclopamine in sheep. METHODS: Five western white-faced ewes were dosed I.V. with purified cyclopamine for pharmacokinetic analysis. Another four groups of five ewes each were dosed orally twice daily with 0.88 g/kg of ground V. californicum root material on either GD 13, 14, or 15 or consecutively on GD days 13-15. Pregnancy and pre-partum fetal malformations were determined by ultrasound imaging on GD 60. At parturition lambs were assessed for gross malformations. RESULTS: The elimination half life of cyclopamine in ewes was determined to be 1.1 ± 0.1 hours. Ewes dosed with ground V. californicum root on GD 13 or 14 had lambs with various craniofacial malformations. Ewes dosed on GD 15 delivered normal lambs. Ewes dosed consecutively on GD 13-15 were all determined to be non-pregnant at 60 days gestation and Veratrum-induced embryonic death was assumed to be the cause. CONCLUSIONS: Cyclopamine is rapidly cleared indicating that ingestion of V. californicum must occur during a very narrow window for teratogenesis to occur. Treatment of pregnant ewes on GD 13 or 14 resulted in synophthalmia and other craniofacial deformities. Lambs with severe malformations, such as cyclopia were smaller, under developed, and appeared premature even though their normal twin counterpart appeared fully developed. Retrospective evaluation suggested that the cyclopic fetuses’ placental development was delayed in comparison to the normal lambs.